Production of adenine nucleotide translocator (ant), novel ant ligands and screening assays therefor

ABSTRACT

Compositions and methods are provided for producing adenine nucleotide translocator (ANT) polypeptides and fusion proteins, including the production and use of recombinant expression constructs having a regulated promoter. ANT ligands and compositions and methods for identifying ANT ligands, agents that bind ANT and agents that interact with ANT are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.09/185,904, filed Nov. 3, 1998.

FIELD OF THE INVENTION

The invention relates to the adenine nucleotide translocator (ANT)protein that is found in mitochondria of eukaryotic cells. Moreparticularly, the invention relates to the production of ANTpolypeptides and ANT fusion proteins using recombinant DNA technology;to novel labeled ligands for ANT proteins; and to assays useful foridentifying and isolating ANT proteins and for screening compounds thatinteract with ANT, including high throughput screening.

BACKGROUND OF THE INVENTION

Mitochondria are the main energy source in cells of higher organisms,and these organelles provide direct and indirect biochemical regulationof a wide array of cellular respiratory, oxidative and metabolicprocesses. These include electron transport chain (ETC) activity, whichdrives oxidative phosphorylation to produce metabolic energy in the formof adenosine triphosphate (ATP), and which also underlies a centralmitochondrial role in intracellular calcium homeostasis.

Mitochondrial ultrastructural characterization reveals the presence ofan outer mitochondrial membrane that serves as an interface between theorganelle and the cytosol, a highly folded inner rnitochondrial membranethat appears to form attachments to the outer membrane at multiplesites, and an intermembrane space between the two mitochondrialmembranes. The subcompartment within the inner mitochondrial membrane iscommonly referred to as the mitochondrial matrix. For a review, see,e.g. Ernster et al., 1981 J. Cell Biol. 91:227s.) The cristae,originally postulated to occur as infoldings of the inner mitochondrialmembrane, have recently been characterized using three-dimensionalelectron tomography as also including tube-like conduits that may formnetworks, and that can be connected to the inner membrane by open,circular (30 nm diameter) junctions (Perkins et al., 1997, Journal ofStructural Biology 119:260). While the outer membrane is freelypermeable to ionic and non-ionic solutes having molecular weights lessthan about ten kilodaltons, the inner mitochondrial membrane exhibitsselective and regulated permeability for many small molecules, includingcertain cations, and is impermeable to large (>˜10 kDa) molecules.

Altered or defective mitochondrial activity, including but not limitedto failure at any step of the ETC, may result in catastrophicmitochondrial collapse that has been termed “permeability transition”(PT) or “mitochondrial permeability transition” (MPT). According togenerally accepted theories of mitochondrial function, proper ETCrespiratory activity requires maintenance of an electrochemicalpotential (ΔΨm) in the inner mitochondrial membrane by a coupledchemiosmotic mechanism. Altered or defective mitochondrial activity maydissipate this membrane potential, thereby preventing ATP biosynthesisand halting the production of a vital biochemical energy source. Inaddition, mitochondrial proteins such as cytochrome c may leak out ofthe mitochondria after permeability transition and may induce thegenetically programmed cell suicide sequence known as apoptosis orprogrammed cell death (PCD).

MPT may result from direct or indirect effects of mitochondrial genes,gene products or related downstream mediator molecules and/orextramitochondrial genes, gene products or related downstream mediators,or from other known or unknown causes. Loss of mitochondrial potentialtherefore may be a critical event in the progression of diseasesassociated with altered mitochondrial function, including degenerativediseases.

Mitochondrial defects, which may include defects related to the discretemitochondrial genome that resides in mitochondrial DNA and/or to theextramitochondrial genome, which includes nuclear chromosomal DNA andother extramitochondrial DNA, may contribute significantly to thepathogenesis of diseases associated with altered mitochondrial function.For example, alterations in the structural and/or functional propertiesof mitochondrial components comprised of subunits encoded directly orindirectly by mitochondrial and/or extramitochondrial DNA, includingalterations deriving from genetic and/or environmental factors oralterations derived from cellular compensatory mechanisms, may play arole in the pathogenesis of any disease associated with alteredmitochondrial function. A number of degenerative diseases are thought tobe caused by, or to be associated with, alterations in mitochondrialfunction. These include Alzheimer's Disease (AD); diabetes mellitus;Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditaryoptic neuropathy; schizophrenia; mitochondrial encephalopathy, lacticacidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferativedisorders; mitochondrial diabetes and deafness (MIDD) and myoclonicepilepsy ragged red fiber syndrome. The extensive list of additionaldiseases associated with altered mitochondrial function continues toexpand as aberrant mitochondrial or mitonuclear activities areimplicated in particular disease processes.

A hallmark pathology of AD and potentially other diseases associatedwith altered mitochondrial function is the death of selected cellularpopulations in particular affected tissues, which results from apoptosis(also referred to as “programmed cell death” or PCD) according to agrowing body of evidence. Mitochondrial dysfunction is thought to becritical in the cascade of events leading to apoptosis in various celltypes (Kroemer et al., FASEB J. 9:1277-87, 1995), and may be a cause ofapoptotic cell death in neurons of the AD brain. Altered mitochondrialphysiology may be among the earliest events in PCD (Zamzami et al., J.Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72,1995) and elevated reactive oxygen species (ROS) levels that result fromsuch altered mitochondrial function may initiate the apoptotic cascade(Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994).

Thus, in addition to their role in energy production in growing cells,mitochondria (or, at least, mitochondrial components) participate inapoptosis (Newmeyer et al., 1994, Cell 79:353-364; Liu et al., 1996,Cell 86:147-157). Apoptosis is apparently also required for, inter alia,normal development of the nervous system and proper functioning of theimmune system. Moreover, some disease states are thought to beassociated with either insufficient (e.g., cancer, autoimmune diseases)or excessive (e.g., stroke damage, AD-associated neurodegeneration)levels of apoptosis. For general reviews of apoptosis, and the role ofmitochondria therein, see Green and Reed (1998, Science 281:1309-1312),Green (1998, Cell 94:695-698) and Kromer (1997, Nature Medicine3:614-620). Hence, agents that effect apoptotic events, including thoseassociated with mitochondrial components, might have a variety ofpalliative, prophylactic and therapeutic uses.

The adenine nucleotide translocator (ANT), a nuclear encoded polypeptidethat is a major component of the inner mitochondrial membrane, isresponsible for mediating transport of ADP and ATP across themitochondrial inner membrane. For example, ANT is believed to mediatestoichiometric ATP/proton exchange across the inner niitochondrialmembrane, and ANT inhibitors (such as atractyloside or bongkrekic acid)induce MPT under certain conditions. Three human ANT isoforms have beendescribed that differ in their tissue expression patterns and othermammalian ANT homologues have been described. (See, e.g., Wallace etal., 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases,Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 283-307,and references cited therein.) ANT has also been implicated as animportant molecular component of the mitochondrial permeabilitytransition pore, a Ca²⁺-regulated inner membrane channel that, asdescribed above, plays an important modulating role in apoptoticprocesses.

As inner mitochondrial membrane proteins are believed to possessmultiple hydrophobic membrane spanning domains, ANT polypeptides mayexhibit, inter alia, poor intracellular solubility, toxic accumulationsand/or the formation of inclusion bodies and other deleterious effectson respiratory homeostasis within a host cell due to ANT biologicalactivity. Consequently, those having ordinary skill in the art haveheretofore been unable to produce ANT reliably or in sufficientquantities for a variety of uses, such as those provided herein. Becauseof the significance of mitochondria to respiratory, metabolic andapoptotic processes, and in view of the prominent role played by ANT inthese and other mitochondrial acitivities, there is clearly a need forcompositions and methods that permit the production of ANT proteins,including ANT fusion proteins; for novel ANT ligands; for methods toidentify and isolate ANT proteins; and for methods of identifying andisolating agents that interact with ANT.

The present invention fulfills these needs and provides other relatedadvantages. These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, various references are set forth belowwhich describe in more detail certain procedures or compositions (e.g.,plasmids, vectors, etc.), and are therefore incorporated by reference intheir entireties.

SUMMARY OF THE INVENTION

In its various aspects and embodiments the invention is directed to:

A recombinant expression construct comprising at least one regulatedpromoter operably linked to a first nucleic acid encoding an adeninenucleotide translocator polypeptide; further comprising at least oneadditional nucleic acid sequence that regulates transcription; whereinthe additional nucleic acid sequence that regulates transcriptionencodes a repressor of said regulated promoter; wherein the adeninenucleotide translocator polypeptide comprises a human adenine nucleotidetranslocator polypeptide; wherein the human adenine nucleotidetranslocator polypeptide is ANT1; wherein the human adenine nucleotidetranslocator polypeptide is ANT2; wherein the human adenine nucleotidetranslocator polypeptide is ANT3; wherein the adenine nucleotidetranslocator polypeptide is expressed as a fusion protein with apolypeptide product of a second nucleic acid sequence; wherein thepolypeptide product of said second nucleic acid sequence is an enzyme;wherein said fusion protein localizes to membranes; wherein saidmembranes are mitochondrial membranes; wherein the adenine nucleotidetranslocator polypeptide is expressed as a fusion protein with at leastone product of a second nucleic acid sequence encoding a polypeptidecleavable by a protease, said adenine nucleotide translocatorpolypeptide being separable from the fusion protein by cleavage with theprotease; A host cell comprising a recombinant expression construct asprovided; wherein the host cell is a prokaryotic cell; wherein the hostcell is a eukaryotic cell; wherein the eukaryotic cell is selected fromthe group consisting of a yeast cell, an insect cell and a mammaliancell; wherein the insect cell is an Sf9 cell or a Trichoplusia ni cell;at lacks at least one isoform of an endogenous adenine nucleotidetranslocator; in which expression of at least one gene encoding anendogenous adenine nucleotide translocator isoform is substantiallyimpaired.

A recombinant expression construct comprising at least one promoteroperably linked to a nucleic acid molecule comprising a first nucleicacid sequence and a second nucleic acid sequence, said first nucleicacid sequence encoding an animal adenine nucleotide translocatorpolypeptide wherein the adenine nucleotide translocator polypeptide isexpressed as a fusion protein with a polypeptide product of said secondnucleic acid sequence; wherein the polypeptide product of said secondnucleic acid sequence is an enzyme; wherein said fusion proteinlocalizes to membranes; wherein said membranes are mitochondrialmembranes; further comprising at least one additional nucleic acidsequence that regulates transcription; wherein the additional nucleicacid sequence that regulates transcription encodes a repressor of saidpromoter; wherein the adenine nucleotide translocator polypeptidecomprises a human adenine nucleotide translocator polypeptide; whereinthe human adenine nucleotide translocator polypeptide is ANT1; whereinthe human adenine nucleotide translocator polypeptide is ANT2; whereinthe human adenine nucleotide translocator polypeptide is ANT3; whereinthe adenine nucleotide translocator polypeptide is expressed as afusion-protein with at least one product of a second nucleic acidsequence encoding a polypeptide cleavable by a protease, said adeninenucleotide translocator polypeptide being separable from the fusionprotein by cleavage with the protease; a host cell comprising arecombinant expression construct as just described; wherein the hostcell is a prokaryotic cell; wherein the host cell is a eukaryotic cell;wherein the eukaryotic cell is selected from the group consisting of ayeast cell, an insect cell and a mammalian cell; wherein the insect cellis an Sf9 cell or a Trichoplusia ni cell; that lacks at least oneisoform of an endogenous adenine nucleotide translocator; in whichexpression of at least one gene encoding an endogenous adeninenucleotide translocator isoform is substantially impaired; wherein theexpression construct is a recombinant viral expression construct;

A method of producing a recombinant adenine nucleotide translocatorpolypeptide, comprising; culturing a host cell comprising a recombinantexpression construct comprising at least one regulated promoter operablylinked to a first nucleic acid encoding an adenine nucleotidetranslocator polypeptide;

A method of producing a recombinant adenine nucleotide translocatorpolypeptide, comprising culturing a host cell comprising a recombinantexpression construct comprising at least one promoter operably linked toa nucleic acid molecule comprising a first nucleic acid sequence and asecond nucleic acid sequence, said first nucleic acid sequence encodingan animal adenine nucleotide translocator polypeptide wherein theadenine nucleotide translocator polypeptide is expressed as a fusionprotein with a polypeptide product of said second nucleic acid sequence;

A method of producing a recombinant adenine nucleotide translocatorpolypeptide, comprising culturing a host cell infected with therecombinant viral expression construct as provided above.

An ANT polypeptide produced by the methods just described.

An isolated human adenine nucleotide translocator polypeptide; whereinthe human adenine nucleotide translocator polypeptide is recombinantANT1 or a variant or fragment thereof; wherein the human adeninenucleotide translocator polypeptide is recombinant ANT2 or a variant orfragment thereof; wherein the human adenine nucleotide translocatorpolypeptide is recombinant ANT3 or a variant or fragment thereof;

An isolated human adenine nucleotide translocator fusion proteincomprising an adenine translocator polypeptide fused to at least oneadditional polypeptide sequence; wherein said one additional polypeptidesequence is an enzyme sequence or a variant or fragment thereof; whereinsaid fusion protein localizes to membranes; wherein said membranes aremitochondrial membranes;

An isolated human adenine nucleotide translocator fusion proteincomprising an adenine translocator polypeptide fused to at least oneadditional polypeptide sequence cleavable by a protease, said adeninenucleotide translocator polypeptide being separable from the fusionprotein by cleavage with the protease.

An isolated adenine nucleotide translocator fusion protein comprising afirst polypeptide that is an animal adenine translocator polypeptidefused to at least one additional polypeptide sequence; wherein said oneadditional polypeptide sequence is an enzyme sequence or a variant orfragment thereof; that localizes to membranes; wherein said membranesare mitochondrial membranes.

An isolated recombinant animal adenine nucleotide translocator fusionprotein comprising an adenine translocator polypeptide fused to at leastone additional polypeptide sequence cleavable by a protease, saidadenine nucleotide translocator polypeptide being separable from thefusion protein by cleavage with the protease; wherein the additionalpolypeptide sequence is a polypeptide having affinity for a ligand.

A method for determining the presence of an ANT polypeptide in abiological sample comprising contacting a biological sample suspected ofcontaining an ANT polypeptide with an ANT ligand under conditions andfor a time sufficient to allow binding of the ANT ligand to an ANTpolypeptide; and detecting the binding of the ANT ligand to an ANTpolypeptide, and therefrom determining the presence of an ANTpolypeptide in said biological sample; wherein the adenine nucleotidetranslocator polypeptide comprises a human adenine nucleotidetranslocator polypeptide; wherein the human adenine nucleotidetranslocator polypeptide is ANT1; wherein the human adenine nucleotidetranslocator polypeptide is ANT2; wherein the human adenine nucleotidetranslocator polypeptide is ANT3; wherein the ANT ligand comprisesatractyloside substituted at 6′ hydroxyl to form an atractylosidederivative; wherein the atractyloside is detectably substituted at the6′ hydroxyl to form a detectable atractyloside derivative; wherein thedetectable atractyloside derivative comprises a radioloabeledsubstituent; wherein the radiolabeled substituent is selected from thegroup consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C and ³⁵S; wherein the detectableatractyloside derivative comprises a fluorescent substituent; whereinthe ANT ligand further comprises a Eu³⁺ atom complexed to theatractyloside derivative; wherein the detectable atractylosidederivative comprises covalently bound biotin; wherein the atractylosidemolecule is substituted at 6′ hydroxyl with an amine or an aminecontaining functionality to form an amine modified atractylosidederivative; wherein the atractyloside molecule is a carboxyatractylosidemolecule that is substituted at 6′ hydroxyl to form an atractylosidederivative that is a carboxyatractyloside derivative.

A method for isolating ANT from a biological sample, comprisingcontacting a biological sample suspected of containing an ANTpolypeptide with an ANT ligand under conditions and for a timesufficient to allow binding of the ANT ligand to an ANT polypeptide; andrecovering the ANT polypeptide, and thereby isolating ANT from abiological sample; wherein the ANT ligand is covalently bound to a solidphase; wherein the ANT ligand is non-covalently bound to a solid phase.

A method for identifying an agent that binds to an ANT polypeptide,comprising contacting a candidate agent with a host cell expressing atleast one recombinant ANT polypeptide under conditions and for a timesufficient to permit binding of the agent to said recombinant ANTpolypeptide; and detecting binding of said agent to the recombinant ANT;wherein the host cell is a prokaryotic cell; wherein the prokaryoticcell is an E. coli cell; wherein the host cell is a eukaryotic cell;wherein the eukaryotic cell is selected from the group consisting of ayeast cell, an insect cell and a mammalian cell; wherein the insect cellis an Sf9 cell or a Trichoplusia ni cell; wherein the host cell lacks atleast one isoform of an endogenous adenine nucleotide translocator;wherein host cell expression of at least one gene encoding an endogenousadenine nucleotide translocator isoform is substantially impaired.

A method for identifying an agent that binds to an ANT polypeptide,comprising contacting a candidate agent with a biological samplecontaining at least one recombinant ANT polypeptide under conditions andfor a time sufficient to permit binding of the agent to said ANTpolypeptide; and detecting binding of said agent to the recombinant ANTpolypeptide.

A method for identifying an agent that interacts with an ANT polypeptidecomprising contacting a biological sample containing recombinant ANTwith a detectable ANT ligand in the presence of a candidate agent; andcomparing binding of the detectable ANT ligand to recombinant ANT in theabsence of said agent to binding of the detectable ANT ligand torecombinant ANT in the presence of said agent, and therefrom identifyingan agent that interacts with an ANT polypeptide.

An ANT ligand comprising atractyloside substituted at the 6′ hydroxyl toform an atractyloside derivative; wherein the atractyloside isdetectably substituted at the 6′ hydroxyl to form a detectableatractyloside derivative; wherein the detectable atractylosidederivative comprises a radioloabeled substituent; wherein theradiolabeled substituent is selected from the group consisting of ¹²⁵I,¹³¹I, ³H, ¹⁴C and ³⁵S; wherein the detectable atractyloside derivativecomprises a fluorescent substituent; further comprising a Eu³⁺ atomcomplexed to the atractyloside derivative; wherein the detectableatractyloside derivative comprises covalently bound biotin; wherein theatractyloside molecule is substituted at 6′ hydroxyl with an amine or anamine containing functionality to form an amine modified atractylosidederivative; wherein the atractyloside molecule is a carboxyatractylosidemolecule that is substituted at 6′ hydroxyl to form an atractylosidederivative that is a carboxyatractyloside derivative.

An ANT ligand having the following structure(I):

including stereoisomers and pharmaceutically acceptable salts thereof,wherein R₁, R₂ and R₃ are as identified below.

An assay plate for high throughput screening of candidate agents thatbind to at least one ANT polypeptide, comprising an assay plate having aplurality of wells, each of said wells further comprising at least oneimmobilized recombinant ANT polypeptide or a variant or fragmentthereof.

A method of targeting a polypeptide of interest to a mitochondrialmembrane, comprising expressing a recombinant expression constructencoding a fusion protein in a host cell, said construct comprising atleast one regulated promoter operably linked to a nucleic acid moleculecomprising a first nucleic acid sequence and a second nucleic acidsequence, said first nucleic acid sequence encoding an adeninenucleotide translocator polypeptide that is expressed as a fusionprotein with a polypeptide product of said second nucleic acid sequence,wherein said second nucleic acid sequence encodes the polypeptide ofinterest.

A method of targeting a polypeptide of interest to a mitochondrialmembrane, comprising expressing a recombinant expression constructencoding a fusion protein in a host cell, said construct comprising atleast one promoter operably linked to a nucleic acid molecule comprisinga first nucleic acid sequence and a second nucleic acid sequence, saidfirst nucleic acid sequence encoding an animal adenine nucleotidetranslocator polypeptide that is expressed as a fusion protein with apolypeptide product of said second nucleic acid sequence, wherein saidsecond nucleic acid sequence encodes the polypeptide of interest; apharmaceutical composition comprising an ANT ligand as just described.

A pharmaceutical composition comprising an agent that binds to an ANTpolypeptide identified as just described. A pharmaceutical compositioncomprising an agent that binds to an ANT polypeptide identified asdescribed above. A pharmaceutical composition comprising an agent thatinteracts with an ANT polypeptide identified above. A method oftreatment comprising administering to a subject any one of the justdescribed the pharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequences of the coding regions of humanANT1 (“ANT1m”), human ANT2 (“ANT2m”) and human ANT3 (“ANT3m”).

FIG. 2 shows the polypeptide sequences of human ANT1 (“ANT1p”), humanANT2 (“ANT2p”) and human ANT3 (“ANT3p”).

FIG. 3 shows induction of His-Tagged, XPRESS™-epitope containg huANT3protein in E. coli as determined by Western analysis.

FIG. 4 shows the localization of His-Tagged, XPRESS™-epitope containghuANT3 protein in E. coli as determined by Western analysis.

FIG. 5 shows the expression of human ANT3 (huANT3) in E. coli expressionsystems.

FIG. 6 shows the expression of huANT3 in baculovirus-infected Sf9 cells.

FIG. 7 shows [³²P]ATP binding to Sf9/huANT3 mitochondria.

FIG. 8 shows that ATP and atractyloside bind competitively to Sf9/huANT3mitochondria.

FIG. 9 shows high-affinity binding of atractyloside to Sf9/huANT3mitochondria.

FIG. 10 shows Northern blot analysis of huANT3 transcripts detected in ayeast expression system. Lane contents: lane “M,” molecular weightmarkers (positions of 1.4, 2.4, 4.4 and 7.5 kilobase markers indicated);lanes 1-3, 10 μg of RNA from three independent isolates of mocktransformed AAC yeast; lanes 4-6, 10 μg of RNA from three independentisolates of AAC yeast transformed with pMK5C (pYPGE2-hANT3); lanes 7-9,10 μg of RNA from three independent isolates of AAC yeast transformedwith pMK5B (pYESTrp2-hANT3); lanes 10 and 11, 0.2 (lane 10) and 0.8(lane 11) μg of RNA prepared from samples of human spleen.

FIG. 11 shows binding of ¹²⁵I-compound 24 to bovine mitochondria.Symbols: (▾), bovine mitochondria; (▪), control (no mitochondria).

FIG. 12 shows binding of ¹²⁵I-compound 24 to mitochondria comprisingrecombinant huANT3. Symbols: (▾), mitochondria from T. ni cellsexpressing huANT3; (▪), control (no mitochondria).

FIG. 13 shows competition of ¹²⁵I-compound 24 binding to bovinemitochondria by unlabeled compound 24(▾), ATR (▪) and ADP (▴).

FIG. 14 shows competition of ¹²⁵I-compound 24 binding to mitochondriafrom T. ni cells expressing huANT3 by unlabeled compound 24 (▾, dashedline), ATR (▪, solid line) and ADP (▴).

FIG. 15 shows competition of ¹²⁵I-compound 24 binding by unlabeled ATRto mitochondria from T. ni cells expressing huANT3 (▴) and control(nontransformed) T. ni cells (♦).

FIG. 16 shows competition of ¹²⁵I-compound 24 binding to beef heartmitochondria by (▪) BKA and (▾) unlabeled compound 24.

FIG. 17 shows competition of ¹²⁵I-compound 24 binding to beef heartmitochondria by compound 23 (▾), compound 28 (♦) and ATR (▪).

FIG. 18 shows competition of ¹²⁵I-compound 24 binding to beef heartmitochondria by compound 5 (♦) and ATR (▪).

FIG. 19 shows competition of ¹²⁵I-compound 24 binding to recombinantHis-tagged huANT3 immobilized on Ni beads by BKA (▴) and ATR (▪).

DETALED DESCRIPTION OF THE INVENTION

The present invention is directed generally toward adenine nucleotidetranslocator (ANT) polypeptides, which as provided herein may refer toany ANT isoform; to expression constructs containing nucleic acidsencoding ANT and to natural and synthetic small molecules that interactwith ANT, including ANT binding ligands. The present invention relatesin part to the unexpected findings that bacterial, insect, yeast ormammalian expression systems can be designed for reliable production ofrecombinant human ANT polypeptides in significant quantities. In certainaspects the invention provides compositions and methods for producingrecombinant ANT polypeptides that employ regulated promoters, and incertain of these and other aspects the invention provides compositionsand methods for producing recombinant ANT polypeptides that are ANTfusion proteins. In certain preferred embodiments, the design of suchexpression systems includes the use of a host cell that lacks endogenousANT or in which endogenous ANT gene expression is substantiallyimpaired, as provided herein.

The present invention thus also pertains in part to methods forproducing and isolating recombinant ANT polypeptides, including humanANT polypeptides and in preferred embodiments human ANT3 polypeptides,that may then be used in various binding assays and screening assays andthe like. In view of the surprising observation that expression ofrecombinant human ANT polypeptides can be achieved at levels enablingsuch uses of these ANT polypeptide products, the present inventionprovides assays (including high throughput assays) for identifyingagents that bind to recombinant human ANT. Accordingly, the presentinvention further relates in part to novel human ANT ligands, thesynthesis, selection and characterization of which would heretofore havenot been possible given the need for expressed recombinant ANTpolypeptides to use in binding assays. The invention also pertains toagents that interact with ANT, including agents that enhance or impairany ANT functions known to the art, including but not limited to thosedescribed herein, and to incorporation of such agents intopharmaceutical compositions and their use in therapeutic methods.

As dicussed above, the present invention relates in part to theunexpected finding that recombinant adenine nucleotide translocator(ANT) polypeptides, which includes full length ANT proteins andpolypeptides, fragments and variants thereof, and further includes ANTfusion proteins as provided herein, can be produced in useful amounts byusing a recombinant expression vector having a regulatory nucleic acidoperably linked to a nucleic acid encoding ANT. In particular, theinvention provides compositions and methods for producing recombinantANT polypeptides through the use of a regulated promoter; the inventionalso provides recombinant ANT polypeptides that are ANT fusion proteins.

The invention also pertains to compositions and methods to identify thepresence of ANT polypeptides and to isolate recombinant ANT, and inaddition to screening assays for compounds that interact with ANT.Accordingly, the invention provides certain advantages with regard toregulation of mitochondrial function, and in particular regulation ofthe mitochondrial permeability “pore”. By way of background, four of thefive multisubunit protein complexes (Complexes I, III, IV and V) thatmediate ETC activity are localized to the inner mitochondrial membrane,which is the most protein rich of biological membranes in cells (75% byweight); the remaining ETC complex (Complex II) is situated in thematrix. ANT represents the most abundant of the inner mitochondrialmembrane proteins. In at least three distinct chemical reactions knownto take place within the ETC, positively-charged protons are moved fromthe mitochondrial matrix, across the inner membrane, to theintermembrane space. This disequilibrium of charged species creates anelectrochemical potential of approximately 220 mV referred to as the“protonmotive force” (PMF), which is often represented by the notationΔψ or Δψm and represents the sum of the electric potential and the pHdifferential across the inner mitochondrial membrane (see, e.g., Ernsteret al., 1981 J. Cell Biol. 91:227s and references cited therein).

This membrane potential drives ANT-mediated stoichiometric exchange ofadenosine triphosphate (ATP) and adenosine diphosphate (ADP) across theinner mitochondrial membrane, and provides the energy contributed to thephosphate bond created when ADP is phosphorylated to yield ATP by ETCComplex V, a process that is “coupled” stoichiometrically with transportof a proton into the matrix. Mitochondrial membrane potential, Δψm, isalso the driving force for the influx of cytosolic Ca²⁺ into themitochondrion. Under normal metabolic conditions, the inner membrane isimpermeable to proton movement from the intermembrane space into thematrix, leaving ETC Complex V as the sole means whereby protons canreturn to the matrix. When, however, the integrity of the innermitochondrial membrane is compromised, as occurs during MPT that mayaccompany a disease associated with altered mitochondrial function,protons are able to bypass the conduit of Complex V without generatingATP, thereby “uncoupling” respiration because electron transfer andassociated proton pumping yields no ATP. Thus, mitochondrialpermeability transition involves the opening of a mitochondrial membrane“pore”, a process by which, inter alla, the ETC and ATP synthesis areuncoupled, Δψm collapses and mitochondrial membranes lose the ability toselectively regulate permeability to solutes both small (e.g., ionicCa²⁺, Na⁺, K⁺, H⁺) and large (e.g., proteins).

Without wishing to be bound by theory, it is unresolved whether thispore is a physically discrete conduit that is formed in mitochondrialmembranes, for example by assembly or aggregation of particularmitochondrial and/or cytosolic proteins and possibly other molecularspecies, or whether the opening of the “pore” may simply represent ageneral increase in the porosity of the mitochondrial membrane.

MPT may also be induced by compounds that bind one or more mitochondrialmolecular components. Such compounds include, but are not limited to,atractyloside and bongkrekic acid, which are known to bind to ANT.Methods of determining appropriate amounts of such compounds to induceMPT are known in the art (see, e.g., Beutner et al., 1998 Biochim.Biophys. Acta 1368:7; Obatomi and Bach, 1996 Toxicol. Lett. 89:155;Green and Reed, 1998 Science 281:1309; Kroemer et al., 1998 Annu. Rev.Physiol. 60:619; and references cited therein). Thus certainmitochondrial molecular components, such as ANT, may contribute to theMPT mechanism. As noted above, it is believed that adenine nucleotidetranslocator (ANT) mediates ATP/proton exchange across the innermitochondrial membrane, and that ANT inhibitors such as atractyloside orbongkrekic acid induce MPT under certain conditions. Hence, it isdesirable to obtain ANT in sufficient quantities for structural andfunctional assays that provide, for example, ANT ligands and otheragents that interact with ANT, which will be useful for therapeuticmanagement of mitochondrial pore activity. See also U.S. Ser. No.09/161,172, entitled “Compositions and Methods for Identifying Agentsthat Alter Mitochondrial Permeability Transition Pores”, which is herebyincorporated by reference.

The compositions and methods of the present invention can be adapted toany prokaryotic or eukaryotic ANT, including plant and animal ANTs,which may further include, for example, yeast, vertebrate, mammalian,rodent, primate and human ANTs, for which amino acid sequences and/orencoding nucleic acids will be known to those familiar with the art.Three human ANT isoforms have been described that differ in their tissueexpression patterns. (Stepien et al., 1992 J. Biol. Chem. 267:14592; seealso Wallace et al., 1998 in Mitochondria & Free Radicals inNeurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds.,Wiley-Liss, New York, pp. 283-307, and references cited therein.)Nucleic acid sequences for cDNAs encoding these three human ANT isoformshave been reported (FIG. 1; See Neckelmann et al., Proc. Nat'l. Acad.Sci. U.S.A. 84:7580-7584 (1987) for huANT1 cDNA [SEQ ID NO:1]; Battiniet al., J. Biol. Chem. 262:4355-4359 (1987) for huANT2 cDNA [SEQ IDNO:2], and Cozens et al., J. Mol. Biol. 206:261-280 (1989) for huANT3cDNA [SEQ ID NO:3]; see FIG. 2 for amino acid sequences of huANT1 [SEQID NO:31] huANT2 [SEQ ID NO:32] and huANT3 [SEQ ID NO:33].), and ANTgene sequences have been determined for a number of species (See, e.g.,Li et al., 1989 J. Biol. Chem. 264:13998 for huANT1 genomic DNA; Liew etal. GenBank Acc. #N86710 for huANT2; Shinohara et al., 1993 Biochim.Biophys. Acta 1152:192 for rat ANT gene; for others see also, e.g., Kuet al., 1990 J. Biol. Chem. 265:16060; Adams et al., 1991 Science252:1651; and WO 98/19714.). ANT sequences among mammalian species arehighly conserved; for example, at the amino acid level murine ANT1 andANT2 exhibit 98% sequence identity with human ANT2. Full length aminoacid sequences of at least 29 ANT proteins have been reported to datefrom a variety of animal and plant species, with most of these deducedfrom nucleic acid sequences. (Fiore et al., 1998 Biochimie 80:137-150)

The present invention further relates to nucleic acids which hybridizeto ANT encoding polynucleotide sequences as provided herein, asincorporated by reference or as will be readily apparent to thosefamiliar with the art, if there is at least 70%, preferably at least90%, and more preferably at least 95% identity between the sequences.The present invention particularly relates to nucleic acids whichhybridize under stringent conditions to the ANT encoding nucleic acidsreferred to herein. As used herein, the term “stringent conditions”means hybridization will occur only if there is at least 95% andpreferably at least 97% identity between the sequences. The nucleicacids which hybridize to ANT encoding nucleic acids referred to herein,in preferred embodiments, encode polypeptides which either retainsubstantially the same biological function or activity as the ANTpolypeptides encoded by the cDNAs of FIG. 1 [SEQ ID NOS:1, 2 and 3], orthe deposited expression constructs.

As used herein, to “hybridize” under conditions of a specifiedstringency is used to describe the stability of hybrids formed betweentwo single-stranded nucleic acid molecules. Stringency of hybridizationis typically expressed in conditions of ionic strength and temperatureat which such hybrids are annealed and washed. Typically “high”,“medium” and “low” stringency encompass the following conditions orequivalent conditions thereto: high stringency: 0.1×SSPE or SSC, 0.1%SDS, 65° C.; medium stringency: 0.2×SSPE or SSC, 0.1% SDS, 50° C.; andlow stringency: 1.0×SSPE or SSC, 0.1% SDS, 50° C.

The deposits referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. § 112. Thesequences of the nucleic acids contained in the deposited materials, aswell as the amino acid sequences of the polypeptides encoded thereby,are incorporated herein by reference and are controlling in the event ofany conflict with any description of sequences herein. A licensee may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

Nucleic Acids

The nucleic acids of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Acoding sequence which encodes an ANT polypeptide for use according tothe invention may be identical to the coding sequence known in the artfor any given ANT, as described above and, for example, as shown forhuman ANT1 [SEQ ID NO:1], human ANT2 [SEQ ID NO:2] and human ANT3 [SEQID NO:3] in FIG. 1, or to that of any deposited clone, or may be adifferent coding sequence, which, as a result of the redundancy ordegeneracy of the genetic code, encodes the same ANT polypeptide as, forexample, the cDNAs of FIG. 1 or the deposited expression constructs.

The nucleic acids which encode ANT polypeptides, for example the humanANT polypeptides having the amino acid sequences of FIG. 2 [SEQ IDNOS:31-33] or any other ANT polypeptides for use according to theinvention, or for the ANT polypeptides encoded by the depositedconstructs may include, but are not limited to: only the coding sequencefor the ANT polypeptide; the coding sequence for the ANT polypeptide andadditional coding sequence; the coding sequence for the ANT polypeptide(and optionally additional coding sequence) and non-coding sequence,such as introns or non-coding sequences 5′ and/or 3′ of the codingsequence for the ANT polypeptide, which for example may further includebut need not be limited to one or more regulatory nucleic acid sequencesthat may be a regulated or regulatable promoter, enhancer, othertranscription regulatory sequence, repressor binding sequence,translation regulatory sequence or any other regulatory nucleic acidsequence. Thus, the term “nucleic acid encoding an ANT polypeptide”encompasses a nucleic acid which includes only coding sequence for thepolypeptide as well as a nucleic acid which includes additional codingand/or non-coding sequence(s).

The present invention further relates to variants of the hereindescribed nucleic acids which encode for fragments, analogs andderivatives of an ANT polypeptide, for example the human ANT1, ANT2 andANT3 polypeptides having the deduced amino acid sequences of FIG. 2 [SEQID NOS:31-33] or any ANT polypeptide, including ANT polypeptides encodedby the cDNAs of the deposited expression constructs. The variants of thenucleic acids encoding ANTs may be naturally occurring allelic variantsof the nucleic acids or non-naturally occurring variants. As is known inthe art, an allelic variant is an alternate form of a nucleic acidsequence which may have at least one of a substitution, a deletion or anaddition of one or more nucleotides, any of which does not substantiallyalter the function of the encoded ANT polypeptide. Thus, for example,the present invention includes nucleic acids encoding the same ANTpolypeptides as shown in FIG. 2 [SEQ ID NOS:31-33], or the same ANTpolypeptides encoded by the cDNAs of the deposited expressionconstructs, as well as variants of such nucleic acids, which variantsencode a fragment, derivative or analog of any of the polypeptides ofFIG. 2 (SEQ ID NO:2) or the polypeptides encoded by the cDNAs of thedeposited expression constructs.

Variants and derivatives of ANT may be obtained by mutations ofnucleotide sequences encoding ANT polypeptides. Alterations of thenative amino acid sequence may be accomplished by any of a number ofconventional methods. Mutations can be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making such alterations are disclosed byWalder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985);Craik (BioTechniques, January 1985, 12-19); Smith et al. (GeneticEngineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc.Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol.154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462.

Equivalent DNA constructs that encode various additions or substitutionsof amino acid residues or sequences, or deletions of terminal orinternal residues or sequences not needed for biological activity arealso encompassed by the invention. For example, sequences encoding Cysresidues that are not essential for biological activity can be alteredto cause the Cys residues to be deleted or replaced with other aminoacids, preventing formation of incorrect intramolecular disulfidebridges upon renaturation. Other equivalents can be prepared bymodification of adjacent dibasic amino acid residues to enhanceexpression in yeast systems in which KEX2 protease activity is present.EP 212,914 discloses the use of site-specific mutagenesis to inactivateKEX2 protease processing sites in a protein. KEX2 protease processingsites are inactivated by deleting, adding or substituting residues toalter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence ofthese adjacent basic residues. Lys-Lys pairings are considerably lesssusceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg toLys-Lys represents a conservative and preferred approach to inactivatingKEX2 sites.

Polypeptides and Fusion Proteins

The present invention further relates to ANT polypeptides, and inparticular to methods for producing recombinant ANT polypeptides byculturing host cells containing ANT expression constructs, and toisolated recombinant human ANT polypeptides, including, for example, thehuman ANT1, ANT2 and ANT3 polypeptides which have the deduced amino acidsequence of FIG. 2 [SEQ ID NOS:31-33] or which have the amino acidsequence encoded by the deposited recombinant expression constructs, aswell as fragments, analogs and derivatives of such polypeptides. Thepolypeptides and nucleic acids of the present invention are preferablyprovided in an isolated form, and in certain preferred embodiments arepurified to homogeneity.

The terms “fragment,” “derivative” and “analog” when referring to ANTpolypeptides or fusion proteins, or to ANT polypeptides or fusionproteins encoded by the deposited recombinant expression constructs,refers to any ANT polypeptide or fusion protein that retains essentiallythe same biological function or activity as such polypeptide. Thus, ananalog includes a proprotein which can be activated by cleavage of theproprotein portion to produce an active ANT polypeptide. The polypeptideof the present invention may be a recombinant polypeptide or a syntheticpolypeptide, and is preferably a recombinant polypeptide.

A fragment, derivative or analog of an ANT polypeptide or fusionprotein, including ANT polypeptides or fusion proteins encoded by thecDNAs of the deposited constructs, may be (i) one in which one or moreof the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe ANT polypeptide is fused with another compound, such as a compoundto increase the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which additional amino acids are fused to theANT polypeptide, including amino acids that are employed forpurification of the ANT polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

The polypeptides of the present invention include ANT polypeptides andfusion proteins having amino acid sequences that are identical orsimilar to sequences known in the art. For example by way ofillustration and not limitation, the human ANT (“huANT”) polypeptides ofFIG. 2 [SEQ ID NOS:31-33] are contemplated for use according to theinstant invention, as are polypeptides having at least 70% similarity(preferably a 70% identity) to the polypeptides of FIG. 2 [SEQ IDNOS:31-33] and more preferably 90% similarity (more preferably a 90%identity) to the polypeptides of FIG. 2 [SEQ ID NOS: 31-33] and stillmore preferably a 95% similarity (still more preferably a 95% identity)to the polypeptides of FIG. 2 [SEQ ID NOS:31-33] and to portions of suchpolypeptides, wherein such portions of an ANT polypeptide generallycontain at least 30 amino acids and more preferably at least 50 aminoacids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide. Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the nucleic acids of the present invention maybe used to synthesize full-length nucleic acids of the presentinvention.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring nucleic acid orpolypeptide present in a living animal is not isolated, but the samenucleic acid or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Such nucleicacids could be part of a vector and/or such nucleic acids orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons).

As described herein, the invention provides ANT fusion proteins encodedby nucleic acids that have the ANT coding sequence fused in frame to anadditional coding sequence to provide for expression of an ANTpolypeptide sequence fused to an additional functional or non-functionalpolypeptide sequence that permits, for example by way of illustrationand not limitation, detection, isolation and/or purification of the ANTfusion protein. Such ANT fusion proteins may permit detection, isolationand/or purification of the ANT fusion protein by protein-proteinaffinity, metal affinity or charge affinity-based polypeptidepurification, or by specific protease cleavage of a fusion proteincontaining a fusion sequence that is cleavable by a protease such thatthe ANT polypeptide is separable from the fusion protein.

Thus ANT fusion proteins may comprise polypeptide sequences added to ANTto facilitate detection and isolation of ANT. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., (1988 Bio/Technology6:1204), or the XPRESS™ epitope tag (Invitrogen, Carlsbad, Calif.). Theaffinity sequence may be a hexa-histidine tag as supplied, for example,by a pBAD/His (Invitrogen) or a pQE-9 vector to provide for purificationof the mature polypeptide fused to the marker in the case of a bacterialhost, or, for example, the affinity sequence may be a hemagglutinin (HA)tag when a mammalian host, e.g., COS-7 cells, is used. The HA tagcorresponds to an antibody defined epitope derived from the influenzahemagglutinin protein (Wilson et al., Cell 37:767 (1984)).

ANT fusion proteins may further comprise immunoglobulin constant regionpolypeptides added to ANT to facilitate detection, isolation and/orlocalization of ANT. The immunoglobulin constant region polypeptidepreferably is fused to the C-terminus of an ANT polypeptide. Generalpreparation of fusion proteins comprising heterologous polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991) and Byrn et al. (Nature 344:677, 1990). A gene fusionencoding the ANT:Fc fusion protein is inserted into an appropriateexpression vector. In certain embodiments of the invention, ANT:Fcfusion proteins may be allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between Fc polypeptides,yielding dimeric ANT fusion proteins.

ANT fusion proteins having specific binding affinities for pre-selectedantigens by virtue of fusion polypeptides comprising immunoglobulinV-region domains encoded by DNA sequences linked in-frame to sequencesencoding ANT are also within the scope of the invention, includingvariants and fragments thereof as provided herein. General strategiesfor the construction of fusion proteins having immunoglobulin V-regionfusion polypeptides are disclosed, for example, in EP 0318554; U.S. Pat.Nos. 5,132,405; 5,091,513; and 5,476,786.

The nucleic acid of the present invention may also encode a fusionprotein comprising an ANT polypeptide fused to other polypeptides havingdesirable affinity properties, for example an enzyme such asglutathione-S-transferase. As another example, ANT fusion proteins mayalso comprise an ANT polypeptide fused to a Staphylococcus aureusprotein A polypeptide; protein A encoding nucleic acids and their use inconstructing fusion proteins having affinity for immunoglobulin constantregions are disclosed generally, for example, in U.S. Pat. No.5,100,788. Other useful affinity polypetides for construction of ANTfusion proteins may include streptavidin fusion proteins, as disclosed,for example, in WO 89/03422; U.S. Pat. No. 5,489,528; U.S. Pat. No.5,672,691; WO 93/24631; U.S. Pat. No. 5,168,049; U.S. Pat. No. 5,272,254and elsewhere, and avidin fusion proteins (see, e.g., EP 511,747). Asprovided herein and in the cited references, ANT polypeptide seqencesmay be fused to fusion polypeptide sequences that may be full lengthfusion polypeptides and that may alternatively be variants or fragmentsthereof.

The present invention also provides a method of targeting a polypeptideof interest to a membrane, and in particular embodiments to a cellularmembrane, and in further embodiments to a mitochondrial membrane. Thisaspect of the invention is based on the unexpected observation thatcertain recombinant expression constructs as provided herein, whichconstructs include a nucleic acid encoding a first polypeptide that isan ANT polypeptide, and that is expressed as a fusion protein with asecond polypeptide sequence, provide recombinant ANT fusion proteinscapable of preferentially localizing to cell membranes. In certainembodiments the cell membrane is a prokaryotic cell membrane such as abacterial cell membrane, for example an E. coli membrane. In otherembodiments the cell membrane is a eukaryotic cell membrane such as ayeast or a mammalian cell membrane, for example a membrane of anyeukaryotic cell contemplated herein.

A cell membrane as used herein may be any cellular membrane, andtypically refers to membranes that are in contact with cytosoliccomponents, including intracellular membrane bounded compartments suchas mitochondrial inner and outer membranes as described above, and alsointracellular vesicles, ER-Golgi constituents, other organelles and thelike, as well as the plasma membrane. In preferred embodiments, an ANTfusion protein may be targeted to a mitochondrial membrane. In otherpreferred embodiments, for example, recombinant expression constructsaccording to the invention may encode ANT fusion proteins that containpolypeptide sequences that direct the fusion protein to be retained inthe cytosol, to reside in the lumen of the endoplasmic reticulum (ER),to be secreted from a cell via the classical ER-Golgi secretory pathway,to be incorporated into the plasma membrane, to associate with aspecific cytoplasmic component including the cytoplasmic domain of atransmembrane cell surface receptor or to be directed to a particularsubcellular location by any of a variety of known intracellular proteinsorting mechanisms with which those skilled in the art will be familiar.Accordingly, these and related embodiments are encompassed by theinstant compositions and methods directed to targeting a polypeptide ofinterest to a predefined intracellular, membrane or extracellularlocalization.

Vectors

The present invention also relates to vectors and to constructs thatinclude nucleic acids of the present invention, and in particular to“recombinant expression constructs” that include any nucleic acidsencoding ANT polypeptides according to the invention as provided above;to host cells which are genetically engineered with vectors and/orconstructs of the invention and to the production of ANT polypeptidesand fusion proteins of the invention, or fragments or variants thereof,by recombinant techniques. ANT proteins can be expressed in mammaliancells, yeast, bacteria, or other cells under the control of appropriatepromoters. Cell-free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989).

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

Useful expression constructs for bacterial use are constructed byinserting into an expression vector a structural DNA sequence encoding adesired protein together with suitable translation initiation andtermination signals in operable reading phase with a functionalpromoter. The construct may comprise one or more phenotypic selectablemarker and an origin of replication to ensure maintenance of the vectorconstruct and, if desirable, to provide amplification within the host.Suitable prokaryotic hosts for transformation include E. coli, Bacillussubtilis, Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus, although others may alsobe employed as a matter of choice. Any other plasmid or vector may beused as long as they are replicable and viable in the host.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter, if itis a regulated promoter as provided herein, is induced by appropriatemeans (e.g., temperature shift or chemical induction) and cells arecultured for an additional period. Cells are typically harvested bycentrifugation, disrupted by physical or chemical means, and theresulting crude extract retained for further purification. Microbialcells employed in expression of proteins can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents; such methods are well know tothose skilled in the art.

Thus, for example, the nucleic acids of the invention as provided hereinmay be included in any one of a variety of expression vector constructsas a recombinant expression construct for expressing an ANT polypeptide.Such vectors and constructs include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA, such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. However, any other vectormay be used for preparation of a recombinant expression construct aslong as it is replicable and viable in the host.

The appropriate DNA sequence(s) may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described, forexample, in Ausubel et al. (1993 Current Protocols in Molecular Biology,Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.);Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring HarborLaboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N.Y.); and elsewhere.

The DNA sequence in the expression vector is operatively linked to atleast one appropriate expression control sequences (e.g., a promoter ora regulated promoter) to direct mRNA synthesis. Representative examplesof such expression control sequences include LTR or SV40 promoter, theE. coli lac or trp, the phage lambda P_(L) promoter and other promotersknown to control expression of genes in prokaryotic or eukaryotic cellsor their viruses. Promoter regions can be selected from any desired geneusing CAT (chloramphenicol transferase) vectors or other vectors withselectable markers. Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lac, lacZ, T3, T7, gpt,lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMV immediateearly, HSV thymidine kinase, early and late SV40, LTRs from retrovirus,and mouse metallothionein-I. Selection of the appropriate vector andpromoter is well within the level of ordinary skill in the art, andpreparation of certain particularly preferred recombinant expressionconstructs comprising at least one promoter or regulated promoteroperably linked to a nucleic acid encoding an ANT polypeptide isdescribed herein.

In certain preferred embodiments the expression control sequence is a“regulated promoter”, which may be a promoter as provided herein and mayalso be a repressor binding site, an activator binding site or any otherregulatory sequence that controls expression of a nucleic acid sequenceas provided herein. In certain particularly preferred embodiments theregulated promoter is a tightly regulated promoter that is specificallyinducible and that permits little or no transcription of nucleic acidsequences under its control in the absence of an induction signal, as isknown to those familiar with the art and described, for example, inGuzinan et al. (1995 J. Bacteriol. 177:4121), Carra et al. (1993 EMBO J.12:35), Mayer (1995 Gene 163:41), Haldimann et al. (1998 J. Bacteriol.180:1277), Lutz et al. (1997 Nuc. Ac. Res. 25:1203), Allgood et al.(1997 Curr. Opin. Biotechnol. 8:474) and Makrides (1996 Microbiol. Rev.60:512), all of which are hereby incorporated by reference. In otherpreferred embodiments of the invention a regulated promoter is presentthat is inducible but that may not be tightly regulated. In certainother preferred embodiments a promoter is present in the recombinantexpression construct of the invention that is not a regulated promoter;such a promoter may include, for example, a constitutive promoter suchas an insect polyhedrin promoter as described in the Examples or a yeastphosphoglycerate kinase promoter (see, e.g., Giraud et al., 1998 J. Mol.Biol. 281:409). The expression construct also contains a ribosomebinding site for translation initiation and a transcription terminator.The vector may also include appropriate sequences for amplifyingexpression.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

As noted above, in certain embodiments the vector may be a viral vectorsuch as a retroviral vector. For example, retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, retrovirusessuch as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The viral vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques 7:980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein, and may be from among either regulated promoters orpromoters as described above.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, 1:5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the ANT polypeptidesor fusion proteins. Such retroviral vector particles then may beemployed, to transduce eukaryotic cells, either in vitro or in vivo. Thetransduced eukaryotic cells will express the nucleic acid sequencers)encoding the ANT polypeptide or fusion protein. Eukaryotic cells whichmay be transduced include, but are not limited to, embryonic stem cells,embryonic carcinoma cells, as well as hematopoietic stem cells,hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells,and bronchial epithelial cells.

As another example of an embodiment of the invention in which a viralvector is used to prepare the recombinant ANT expression construct, inone preferred embodiment, host cells transduced by a recombinant viralconstruct directing the expression of ANT polypeptides or fusionproteins may produce viral particles containing expressed ANTpolypeptides or fusion proteins that are derived from portions of a hostcell membrane incorporated by the viral particles during viral budding.In another preferred embodiment, ANT encoding nucleic acid sequences arecloned into a baculovirus shuttle vector, which is then recombined witha baculovirus to generate a recombinant baculovirus expression constructthat is used to infect, for example, Sf9 or Trichoplusia ni (PharMingen,Inc., San Diego, Calif.) host cells, as described in BaculovirusExpression Protocols, Methods in Molecular Biology Vol. 39, ChristopherD. Richardson, Editor, Human Press, Totowa, N.J., 1995; Piwnica-Worms,“Expression of Proteins in Insect Cells Using Baculoviral Vectors,”Section II in Chapter 16 in: Short Protocols in Molecular Biology,2^(nd) Ed., Ausubel et al., eds., John Wiley & Sons, New York, N.Y.,1992, pages 16-32 to 16-48.

Host Cells

In another aspect, the present invention relates to host cellscontaining the above described recombinant ANT expression constructs.Host cells are genetically engineered (transduced, transformed ortransfected) with the vectors and/or expression constructs of thisinvention which may be, for example, a cloning vector, a shuttle vectoror an expression construct. The vector or construct may be, for example,in the form of a plasmid, a viral particle, a phage, etc. The engineeredhost cells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying particular genes such as genes encoding ANT polypeptides orANT fusion proteins. The culture conditions for particular host cellsselected for expression, such as temperature, pH and the like, will bereadily apparent to the ordinarily skilled artisan.

The host cell can be a higher eukaryotic cell, such as a mammalian cell,or a lower eukaryotic cell, such as a yeast cell, or the host cell canbe a prokaryotic cell, such as a bacterial cell. Representative examplesof appropriate host cells according to the present invention include,but need not be limited to, bacterial cells, such as E. coli,Streptomyces, Salmonella tvphimurium; fungal cells, such as yeast;insect cells, such as Drosophila S2, Trichoplusia ni (PharMingen, SanDiego, Calif.) and Spodoptera Sf9; animal cells, such as CHO, COS or 293cells; adenoviruses; plant cells, or any suitable cell already adaptedto in vitro propagation or so established de novo. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences, for example as described herein regarding the preparation ofANT expression constructs. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements. Introduction of the construct into thehost cell can be effected by a variety of methods with which thoseskilled in the art will be familiar, including but not limited to, forexample, calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis et al., 1986 Baic Methods inMolecular Biology).

As will be aprreciated by those of ordinary skill in the art, in certainsituations it may be desirable to prepare the compositions of theinvention and to practice the methods of the invention under conditionswhere endogenous ANT expression by a host cell is compromised, in orderto provide advantages associated with the expression of a desired ANTencoding construct. For example, detection of particular ANT encodingnucleic acid sequences or ANT polypeptides that are highly similar tothose encoded by the host cell genome may be facilitated by inhibitinghost cell ANT gene expression. As another example, where functionalactivity of an exogenously introduced recombinant ANT polypeptide is tobe determined in a host cell or in a biological sample derivedtherefrom, it may also be advantageous to inhibit endogenous host cellANT gene expression.

Thus, in certain preferred embodiments of the invention, host cells maylack at least one isofonn of an endogenous ANT, and in certain preferredembodiments the host cells may lack all endogenous ANT isoforms. Forexample, in the yeast system described by Giraud et al. (1998 J. Mol.Biol. 281:409) a S. cerevisiae triple null mutant is described thatlacks all three yeast ANT isoforms and is unable to grow under anaerobicconditions. In other preferred embodiments, expression in host cells ofat least one gene encoding an endogenous ANT isoform is substantiallyimpaired. Substantial impairment of endogenous ANT isoform expressionmay be achieved by any of a variety of methods that are well known inthe art for blocking specific gene expression, including site-specificor site-directed mutagenesis as described above, antisense inhibition ofgene expression, ribozyme mediated inhibition of gene expression andgeneration of mitochondrial DNA depleted (ρ⁰) cells.

Identification of oligonucleotides and ribozymes for use as antisenseagents and DNA encoding genes for targeted delivery for genetic therapyinvolve methods well known in the art. For example, the desirableproperties, lengths and other characteristics of such oligonucleotidesare well known. Antisense oligonucleotides are typically designed toresist degradation by endogenous nucleolytic enzymes by using suchlinkages as: phosphorothioate, methylphosphonate, sulfone, sulfate,ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and othersuch linkages (see, e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542(1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec etal., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. AcidsRes. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989);Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev.Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100(1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of GeneExpression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jageret al., Biochemistry 27:7237-7246 (1988)).

Antisense nucleotides are oligonucleotides that bind in asequence-specific manner to nucleic acids, such as mRNA or DNA. Whenbound to mRNA that has complementary sequences, antisense preventstranslation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053 to Altman etal.; U.S. Pat. No. 5,190,931 to Inouye, U.S. Pat. No. 5,135,917 toBurch; U.S. Pat. No. 5,087,617 to Smith and Clusel et al. (1993) Nucl.Acids Res. 21:3405-3411, which describes dumbbell antisenseoligonucleotides). Triplex molecules refer to single DNA strands thatbind duplex DNA forming a colinear triplex molecule, thereby preventingtranscription (see, e.g., U.S. Pat. No. 5,176,996 to Hogan et al., whichdescribes methods for making synthetic oligonucleotides that bind totarget sites on duplex DNA).

According to this embodiment of the invention, particularly usefulantisense nucleotides and triplex molecules are molecules that arecomplementary to or bind the sense strand of DNA or mRNA that encodes anANT polypeptide or a protein mediating any other process related toexpression of endogenous ANT genes, such that inhibition of translationof mRNA encoding the ANT polypeptide is effected.

A ribozyme is an RNA molecule that specifically cleaves RNA substrates,such as mRNA, resulting in specific inhibition or interference withcellular gene expression. There are at least five known classes ofribozymes involved in the cleavage and/or ligation of RNA chainsRibozymes can be targeted to any RNA transcript and can catalyticallycleave such transcripts (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat.No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and5,093,246 to Cech et al.). According to certain embodiments of theinvention, any such ANT mRNA-specific ribozyme, or a nucleic acidencoding such a ribozyme, may be delivered to a host cell to effectinhibition of ANT gene expression. Ribozymes, and the like may thereforebe delivered to the host cells by DNA encoding the ribozyme linked to aeukaryotic promoter, such as a eukaryotic viral promoter, such that uponintroduction into the nucleus, the ribozyme will be directlytranscribed.

As used herein, expression of a gene encoding an endogenous adeninenucleotide translocator isoform is substantially impaired by any of theabove methods for inhibiting when cells are substantially but notnecessarily completely depleted of functional DNA or functional mRNAencoding the endogenous ANT isoform, or of the relevant ANT polypeptide.ANT isoform expression is substantially impaired when cells arepreferably at least 50% depleted of DNA or mRNA encoding the endogenousANT (as measured using high stringency hybridization as described above)or depleted of ANT polypeptide (as measured by Western immunoblot asdescribed herein, see also, e.g., Giraud et al. 1998 J. Mol Biol.281:409); and more preferably at least 75% depleted of endogenous ANTDNA, mRNA or polypeptide. Most preferably, ANT isoform expression issubstantially impaired when cells are depleted of >90% of theirendogenous ANT DNA, mRNA, or polypeptide.

Alternatively, expression of a gene encoding an endogenous adeninenucleotide translocator isoform may be substantially impaired throughthe use of mitochondrial DNA depleted ρ⁰ cells, which are incapable ofmitochondrial replication and so may not continue to express functionalANT polypeptides. Methods for producing ρ⁰ cells are known and can befound, for example in PCT/US95/04063, which is hereby incorporated byreference.

Protein Production

The expressed recombinant ANT polypeptides or fusion proteins may beuseful in intact host cells; in intact organelles such as mitochondria,cell membranes, intracellular vesicles other cellular organelles; or indisrupted cell preparations including but not limited to cellhomogenates or lysates, submitochondrial particles, uni- andmultilamellar membrane vesicles or other preparations. Alternatively,expressed recombinant ANT polypeptides or fusion proteins can berecovered and purified from recombinant cell cultures by methodsincluding ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by acterial, yeast, higher plant, insect and mammalian cells inculture). Depending upon the host employed in a recombinant productionprocedure, the polypeptides of the present invention may be glycosylatedor may be non-glycosylated. Polypeptides of the invention may alsoinclude an initial methionine amino acid residue.

Samples

A “biological sample containing mitochondria” may comprise any tissue orcell preparation in which intact mitochondria capable of maintaining amembrane potential when supplied with one or more oxidizable substratessuch as glucose, malate or galactose are or are thought to be present.Mitochondrial membrane potential may be determined according to methodswith which those skilled in the art will be readily familiar, includingbut not limited to detection and/or measurement of detectable compoundssuch as fluorescent indicators, optical probes and/or sensitive pH andion-selective electrodes (See, e.g., Ernster et al., 1981 J. Cell Biol.91:227s and references cited therein; see also Haugland, 1996 Handbookof Fluorescent Probes and Research Chemicals—Sixth Ed., MolecularProbes, Eugene, Oreg., pp. 266-274 and 589-594.). By “capable ofmaintaining a potential” it is meant that such mitochondria have amembrane potential that is sufficient to permit the accumulation of adetectable compound (e.g., DASPMI[2-,4-dimethylaminostyryl-N-methylpyridinium], TMRM[tetramethylrhodamine methyl ester], etc.) used in the particularinstance. A biological sample containing mitochondria may, for example,be derived from a normal (i.e., healthy) individual or from anindividual having a disease associated with altered mitochondrialfunction. Biological samples containing mitochondria may be provided byobtaining a blood sample, biopsy specimen, tissue explant, organ cultureor any other tissue or cell preparation from a subject or a biologicalsource. The subject or biological source may be a human or non-humananimal, a primary cell culture or culture adapted cell line includingbut not limited to genetically engineered cell lines that may containchromosomally integrated or episomal recombinant nucleic acid sequences,immortalized or immortalizable cell lines, somatic cell hybrid orcytoplasmic hybrid “cybrid” cell lines, differentiated ordifferentiatable cell lines, transformed cell lines and the like.

A “biological sample” may comprise any tissue or cell preparation asjust described for a biological sample containing mitochondria, but doesnot require the presence of intact mitochondria. Thus a “biologicalsample” may comprise any tissue or cell preparation and a “biologicalsample containing at least one recombinant ANT polypeptide” comprisesany tissue or cell preparation in which an expressed recombinant ANTpolypeptide or fusion protein as provided herein is thought to bepresent. A biological sample may, for example, be derived from arecombinant cell line or from a transgenic animal. Biological samplescontaining recombinant ANT may be provided by obtaining a blood sample,biopsy specimen, tissue explant, organ culture or any other tissue orcell preparation from a subject or a biological source. The subject orbiological source may be a human or non-human animal, a primary cellculture or culture adapted cell line including but not limited togenetically engineered cell lines that may contain chromosomallyintegrated or episomal recombinant nucleic acid sequences, immortalizedor immortalizable cell lines, somatic cell hybrid or cytoplasmic hybrid“cybrid” cell lines, differentiated or differentiatable cell lines,transformed cell lines and the like.

Proteins

As described herein, isolation of a mitochondrial pore component or amitochondrial molecular species with which an agent identified accordingto the methods of the invention interacts refers to physical separationof such a complex from its biological source, and may be accomplished byany of a number of well known techniques including but not limited tothose described herein, and in the cited references. Without wishing tobe bound by theory, a compound that “binds a mitochondrial component”can be any discrete molecule, agent compound, composition of matter orthe like that may, but need not, directly bind to a mitochondrialmolecular component, and may in the alternative bind indirectly to amitochondrial molecular component by interacting with one or moreadditional components that bind to a mitochondrial molecular component.These or other mechanisms by which a compound may bind to and/orassociate with a mitochondrial molecular component are within the scopeof the claimed methods, so long as isolating a mitochondrial porecomponent also results in isolation of the mitochondrial molecularspecies that directly or indirectly binds to the identified agent. Thus,for example, as provided herein, any ANT polypeptide includingrecombinant ANT polypeptides and fusion proteins may be a mitochondrialmolecular component and/or a mitochondrial pore component, and any ANTligand or agent that binds to an ANT polypeptide may be a compound thatbinds a mitochondrial component and/or an agent that affectsmitochondrial pore activity.

As described herein, the mitochondrial permeability transition “pore”may not be a discrete assembly or multisubunit complex, and the termthus refers instead to any mitochondrial molecular component (including,e.g., a mitochondrial membrane per se) that regulates the inner membraneselective permeability where such regulated function is impaired duringMPT. As used herein, mitochondria are comprised of “mitochondrialmolecular components”, which may be any protein, polypeptide, peptide,amino acid, or derivative thereof, any lipid, fatty acid or the like, orderivative thereof; any carbohydrate, saccharide or the like orderivative thereof, any nucleic acid, nucleotide, nucleoside, purine,pyrimidine or related molecule, or derivative thereof, or the like; orany other biological molecule that is a constituent of a mitochondrion.“Mitochondrial molecular components” includes but is not limited to“mitochondrial pore components”. A “mitochondrial pore component” is anymitochondrial molecular component that regulates the selectivepermeability characteristic of mitochondrial membranes as describedabove, including those responsible for establishing ATm and those thatare functionally altered during MPT.

Isolation and, optionally, identification and/or characterization of themitochondrial pore component or components with which an agent thataffects mitochondrial pore activity interacts may also be desirable andare within the scope of the invention. Once an agent is shown to alterMPT according to the methods provided herein and in U.S. Ser. No.09/161,172, those having ordinary skill in the art will be familiar witha variety of approaches that may be routinely employed to isolate themolecular species specifically recognized by such an agent and involvedin regulation of MPT, where to “isolate” as used herein refers toseparation of such molecular species from the natural biologicalenvironment. Thus, for example, once an ANT ligand is prepared accordingto the methods provided herein, such approaches may be routinelyemployed to isolate the ANT polypeptide. Techniques for isolating amitochondrial pore component such as an ANT polypeptide or fusionprotein may include any biological and/or biochemical methods useful forseparating the complex from its biological source, and subsequentcharacterization may be performed according to standard biochemical andmolecular biology procedures. Those familiar with the art will be ableto select an appropriate method depending on the biological startingmaterial and other factors. Such methods may include, but need not belimited to, radiolabeling or otherwise detectably labeling cellular andmitochondrial components in a biological sample, cell fractionation,density sedimentation, differential extraction, salt precipitation,ultrafiltration, gel filtration, ion-exchange chromatography, partitionchromatography, hydrophobic chromatography, electrophoresis, affinitytechniques or any other suitable separation method that can be adaptedfor use with the agent with which the mitochondrial pore componentinteracts. Antibodies to partially purified components may be developedaccording to methods known in the art and may be used to detect and/orto isolate such components.

Affinity techniques may be particularly useful in the context of thepresent invention, and may include any method that exploits a specificbinding interaction between a mitochondrial pore component and an agentidentified according to the invention that interacts with the porecomponent. For example, because ANT ligands as provided herein and otheragents that influence MPT can be immobilized on solid phase matrices, anaffinity binding technique for isolation of the pore component may beparticularly useful. Alternatively, affinity labeling methods forbiological molecules, in which a known MPT-active agent or a novel ANTligand as provided herein may be modified with a reactive moiety, arewell known and can be readily adapted to the interaction between theagent and a pore component, for purposes of introducing into the porecomponent a detectable and/or recoverable labeling moiety. (See, e.g.,Pierce Catalog and Handbook, 1994 Pierce Chemical Company, Rockford,Ill.; Scopes, R. K., Protein Purification: Principles and Practice,1987, Springer-Verlag, New York; and Hermanson, G. T. et al.,Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc.,California; for details regarding techniques for isolating andcharacterizing biological molecules, including affinity techniques.

Characterization of mitochondrial pore component molecular species,isolated by MPT-active agent affinity techniques described above or byother biochemical methods, may be accomplished using physicochemicalproperties of the pore component such as spectrometric absorbance,molecular size and/or charge, solubility, peptide mapping, sequenceanalysis and the like. (See, e.g., Scopes, supra.) Additional separationsteps for biomolecules may be optionally employed to further separateand identify molecular species that co-purify with mitochondrial porecomponents. These are well known in the art and may include anyseparation methodology for the isolation of proteins, lipids, nucleicacids or carbohydrates, typically based on physicochemical properties ofthe newly identified components of the complex. Examples of such methodsinclude RP-HPLC, ion exchange chromatography, hydrophobic interactionchromatography, hydroxyapatite chromatography, native and/or denaturingone- and two-dimensional electrophoresis, ultrafiltration, capillaryelectrophoresis, substrate affinity chromatography, immunoaffinitychromatography, partition chromatography or any other useful separationmethod. Preferably extracts of cultured cells, and in particularlypreferred embodiments extracts of biological tissues or organs may besources of mitochondrial molecular components, including ANTpolypeptides. Preferred sources may include blood, brain, fibroblasts,myoblasts, liver cells or other cell types.

ANT Ligands

As noted above, the binding of the adenine nucleotide translocator (ANT)is responsible for mediating transport of ADP and ATP across themitochondrial inner membrane. ANT has also been implicated as thecritical component of the mitochondrial permeability transition pore, aCa²⁺ regulated inner membrane channel that plays an important modulatingrole in apoptotic processes. Additionally, ANT activity appears to berelated to changes in ANT polypeptide conformation within themitochondrial membrane, as evidenced by studies using agents that arecapable of binding to ANT. (Block et al., 1986 Meths. Enzymol. 125:658)Accordingly, it is another aspect of the present invention to providecompositions and methods for producing and identifying agents that bindto ANT, which agents are also referred to herein as ANT ligands.

Binding interactions between ANT and a variety of small molecules areknown to those familiar with the art. For example, these interactionsinclude binding to ANT by atractyloside, carboxyatractyloside,palmitoyl-CoA, bongkrekic acid, thyroxin, eosin Y and erythrosin B.(See, e.g., Stubbs, 1979 Pharm. Ther. 7:329; Klingenberg et al., 1978Biochim. Biophys. Acta 503:193; Sterling, 1986 Endocrinol. 119:292;Majima et al., 1998 Biochem. 37:424; Block et al. 1986 Meths. Enzymol.125:658; for erythrosin B and additional ANT inhibitors, see Beavis etal. 1993 J. Biol. Chem. 268:997; Powers et al. 1994 J. Biol. Chem.269:10614.)

The ANT ligands of the present invention represent novel atractylosidederivatives. Atractyloside (ATR) and its known derivatives, includingcarboxyatractyloside (CATR), naphthoyl-ATR, MANT-ATR and other ATRderivatives (see, e.g., Boulay et al., Analytical Biochemistry128:323-330,1983; Roux et al., Analytical Biochemistry 234:31-37,1996;Lauquin et al., FEBS Letters 67:306-311,1976; and Gottikh et al.,Tetrahedron 26:4419-4433, 1970; for other known ATR derivatives see,e.g., Block et al., 1986 Meths. Enzymol. 125:658) have proven invaluablein the elucidation of the structure and the mechanism of action of theadenine nucleotide translocator. According to the ANT ligands of theinvention, the binding mode of ATR to ANT allows for modifications ofthe ATR 6′-hydroxyl functionality without significantly altering ATRbinding affinity for ANT. Thus, ANT ligands as provided herein may beATR derivatives modified by chemical substitution at the 6′ hydroxylposition. In particular, the novel ANT ligands as provided hereinfurther include long linker moieties at the 6′ position, which linkersmay include a 6′-amine linker, thereby permitting additional chemicalmodification to the ANT ligand as will be appreciated by those skilledin the art and as illustrated in the non-limiting Examples. Also, asshown in Examples 6-11, such linkers as provided herein may have carbonchain backbones of 2-20 carbon atoms, and in preferred embodiments 2-6carbon atoms.

The invention therefore provides ANT ligands that may be intermediatesfor conjugation to a variety of additional chemical moieties to yieldfurther ATR derivatives that are ANT ligands within the scope of theinvention. These include ANT ligands to which ¹²⁵I may be covalentlyattached under mild reaction conditions; the invention also includes ANTligands to which reactive amine groups may be covalently linked. ANTligands which are such amine-containing ATR derivatives may then bereacted with a variety of fluorophores and haptens bearing, for example,reactive isothiocyanate, N-hydroxysuccinimide ester, anhydride and otheruseful functionalities to yield stable ATR derivatives including, forexample, derivatives that have thiourea, amide or other linkages.

Thus, ANT ligands as provided herein also include ATR derivatives thatare detectable by virtue of substituents introduced at the 6′ position.Accordingly, detectable ATR derivatives as herein provided include ATRderivatives having a 6′ hydroxyl substitution that includes aradiolabeled substituent, for example ¹²⁵I, ¹³¹I, ³H, ¹⁴C or ³⁵S. OtherANT ligands that are detectable AIR derivatives may comprise fluorescentsubstituents, including those appropriately tagged with reportermolecules such as fluorophores and haptens having utility in highthroughput screening assays for identifying agents that bind to ANT.More specifically, in preferred embodiments, an ANT ligand according tothe present invention that includes fluorescent substituents has anextinction coefficient ≧10,000 M⁻¹ (see Table 1); further, this propertyprovides an advantage for using such ANT ligands according to themethods provided herein, and in particular for use in high throughputscreening assays. Additionally, the ANT ligands of the invention exhibithigh affinities for ANT, and in preferred embodiments have bindingconstants in the nanomolar range.

In certain embodiments of the invention, ANT ligands may be ATRderivatives such as ATR-lanthanide chelating agents, which have utilityin time-resolved fluorescence detection, for example detection of thesecompounds complexed to a lanthanide ion such as Eu³⁺, Tb³⁺, Sm³⁺ andDy³⁺. In addition, ANT ligands may comprise ATR conjugated to readilydetectable substituents such as highly fluorescent moieties, for exampleby way of illustration and not limitation, cyanine and coumarinderivatives. These and other highly fluorescent substituents permit thesynthesis, according to the methods of the invention, of ANT ligandsthat are detectable with extremely high sensitivities. Those familiarwith the art are aware of additional fluorescent substituents that maybe used, for example, those disclosed in Haugland, 1996 Handbook ofFluorescent Probes and Research Chemicals—Sixth Ed., Molecular Probes,Eugene, Oreg. In other embodiments, the invention provides detectableANT ligands produced by coupling of biotin-NHS ester with the ATRderivatives as disclosed herein; these and other ANT ligands similarlygenerated according to the instant methods can be detected withcommercially available enzyme-avidin conjugates using, for example,colorimetric, fluorescent or chemiluminescent techniques.

In one embodiment, the ANT ligands of this invention have the followingstructure(I):

including stereoisomers and pharmaceutically acceptable salts thereof,

-   -   wherein        -   R₁ is hydroxyl, halogen, —OC(═O)R₄ or —NHR₄;        -   R₂ is hydrogen or —C(═O)R₅;        -   R₃ is —CH₃ or ═CH₂;        -   R₄ is -X-aryl, -X-substituted aryl, -X-arylalkyl,            -X-substituted arylalkyl, X-heteroaryl, or            -X-heteroarylalkyl, wherein X is an optional amido or            alkylamido linker moiety; and        -   R₅ is alkyl.

As used herein, the above terms have the meanings set forth below.

“Amido” means —NHC(═O)— or —C(═O)NH—.

“Alkylamido” means -(alkyl)-NHC(═O)— or -(alkyl)-C(═O)NH—, such as—CH₂NHC(═O)—, —CH₂CH₂NHC(═O)—, —CH₂C(═O)NH—, —CH₂CH₂C(═O)NH—, and thelike.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,unsaturated or saturated aliphatic hydrocarbon containing from 1 to 8carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; whilesaturated branched alkyls include isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, and the like. Representative saturated cyclicalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and thelike; while unsaturated cyclic alkyls include cyclopentenyl andcyclohexenyl, and the like. Unsaturated alkyls contain at least onedouble or triple bond between adjacent carbon atoms (referred to as an“alkenyl” or “alkynyl”, respectively). Representative straight chain andbranched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; whilerepresentative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Aryl” means an aromatic carbocyclic moiety such as phenyl or naphthyl(i.e., 1- or 2-naphthyl).

“Arylalkyl” means an alkyl having at least one alkyl hydrogen atomsreplaced with an aryl moiety, such as benzyl, —(CH₂)₂phenyl,—(CH₂)₃phenyl, and the like.

“Heteroaryl” means an aromatic heterocycle ring of 5- to 10 members andhaving at least one heteroatom selected from nitrogen, oxygen andsulfur, and containing at least 1 carbon atom, including both mono- andbicyclic ring systems. Representative heteroaryls are pyridyl, furyl,benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl,indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl,benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, andquinazolinyl.

“Heteroarylalkyl” means an alkyl having at least one alkyl hydrogen atomreplaced with a heteroaryl moiety, such as —CH₂pyridinyl,—CH₂pyrimidinyl, and the like.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 to 4 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined above. Thus, in addition tothe heteroaryls listed above, heterocycles also include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

“Heterocyclealkyl” means an alkyl having at least one alkyl hydrogenatom replaced with a heterocycle, such as —CH₂morpholinyl, and the like.

The term “substituted” as used herein means any of the above groups(i.e., alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycleand heterocyclealkyl) wherein at least one hydrogen atom is replacedwith a substituent. In the case of a keto substituent (“C(═O)”) twohydrogen atoms are replaced. Substituents include halogen, hydroxy,alkyl, haloalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heterocycle, substitued heterocycle, heterocyclealkyl orsubstituted heterocyclealkyl.

“Halogen” means fluoro, chloro, bromo and iodo.

“Haloalkyl” means an alkyl having at least one hydrogen atom replacedwith halogen, such as trifluoromethyl and the like.

“Alkoxy” means an alkyl moiety attached through an oxygen bridge (i.e.,—O-alkyl) such as methoxy, ethoxy, and the like.

In one embodiment, R₂ is —C(═O)CH₂CH(CH₃)₂, R₃ is ═CH₂, and the ANTligand is an atractyloside derivative having the following structure(II):

wherein R₁ is as defined above.

In another embodiment, R₂ is —C(═O)CH₂CH(CH₃)₂, R₃ is —CH₃, and the ANTligand is a dihydro-atractyloside derivative having the followingstructure (III):

wherein R₁ is as defined above.

In still a further embodiment, R₂ is —OH, R₃ is ═CH₂, and the ANT ligandis an apoatractyloside derivative having the following structure (IV):

wherein R₁ is as defined above.

In more specific embodiments of structures (II), (III) and (IV), R₁ is—OC(═O)(aryl), —OC(═O)(substituted aryl), —OC(═O)(arylalkyl),—OC(═O)(substituted arylalkyl), —NH(CH₂)₂NHC(═O)(arylalkyl),—NH(CH₂)₂NHC(═O)(substituted arylalkyl). Representative R₁ moieties inthis regard include —OC(═O)(phenyl), —OC(═O)(1-naphthyl),—OC(═O)(substituted phenyl), —OC(═O)(substituted 1-naphthyl),—OC(═O)(CH₂)₁₋₃(phenyl), —OC(═O)(CH₂)₁₋₃(substituted phenyl),—NH(CH₂)₂NHC(═O)(CH₂)₁₋₃(phenyl), —NH(CH₂)₂NHC(═O)(CH₂)₁₋₃(substitutedphenyl). In this context, representative substituted phenyl moietiesinclude (but are not limited to) 4-hydroxyphenyl,3-iodo-4-hydroxyphenyl, 3,5-iodo-4-hydroxyphenyl,4-(4-hydoxyphenyl)phenyl, 4-(3-iodo-4-hyroxyphenyl)phenyl,3-methyl-4-hyroxyphenyl, and 3-methyl-4-hydroxy-5-iodophenyl.

The ANT ligands of structure (I) may readily be made by one skilled inthe art of organic chemisty and, more particularly, by the techniquesdisclosed in Examples 6-11.

The compounds of the present invention may generally be utilized as thefree base. Alternatively, the compounds of this invention may be used inthe form of acid addition salts. Acid addition salts of the free aminocompounds of the present invention may be prepared by methods well knownin the art, and may be formed from organic and inorganic acids. Suitableorganic acids include maleic, fumaric, benzoic, ascorbic, succinic,methanesulfonic, acetic, oxalic, propionic, tartaric, salicylic, citric,gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic,glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acidsinclude hydrochloric, hydrobromic, sulfiric, phosphoric, and nitricacids. Thus, the term “pharmaceutically acceptable salt” of structure(I) is intended to encompass any and all acceptable salt forms.

With regard to stereoisomers, the compounds of structure (I) may havechiral centers and may occur as recemates, reacemic mixtures and asindividual enantiomers or diastereomers. All such isomeric forms areincluded within the present invention, including mixtures thereof.Furthermore, some of the crystalline forms of the compounds of structure(I) may exist as polymorphs, which are included in the presentinvention. In addition, some of the compounds of structure (I) may alsoform solvates with water or other organic solvents. Such solvates aresimilarly included within the scope of this invention.

Activities of ANT ligands are typically calculated from the IC₅₀ as theconcentration of a compound necessary to displace 50% of the detectable(i.e., detectably labeled, for example, radiolabeled) ligand from ANTmolecules, which may be present as isolated or purified polypeptides oras components of preparations containing isolated mitochondria orsubmitochondrial particles (SMP) using established ligand binding assaysor modifications thereof For example, ANT ligands may be tested fortheir ability to compete with radiolabeled ATR, or with a radiolabeledATR derivative such as compound 24 as provided herein, for binding toisolated ANT polypeptides or to ANT present in isolated mitochondria orSMP.

As another example, the relative affinities for ANT of various ANTligands as provided herein may be determined by a fluorescence assaythat exploits the flourescent properties of compound 22 (Example 11), anaphthoyl-ATR derivative that is an ANT ligand having a fluorescenceexcitation peak at 312 nm and an emission peak at 400 nm. When compound22 is bound to ANT, the fluorescence is quenched. When, however,compound 22 is displaced from ANT by a known concentration of ATR or anATR derivative that is an ANT ligand, fluorescence dequenching thatresults from displacement of the fluorophore can be measured in realtime.

Briefly, a mitochondrial preparation (see, e.g. Example 13) is washedand resuspended in a suitable buffer in the presence of compound 22(e.g., 10 mM Tris-120 mM KCl containing 3.6 nmoles of compound 22 per mgmitochondrial protein, 10 min at room temperature), washed to removeunbound fluorophore and placed in a fluorometer equipped with a lightsource and filter set appropriate for the fluorophore. Fluorescenceintensity is monitored as a function of time, and a candidate ANT ligandis then added to determine its ability to compete with compound 22 forbinding to ANT, as evidenced by a change in detectable relativefluorescence intensity units. After the fluorescence signal hasstabilized, any additional compound 22 that remains bound to ANT isdisplaced by adding an excess (e.g., μM quantities) of ATR as acompetitive inhibitor, to determine maximal signal intensity andtherefrom calculate the proportion of compound 22 displaced by thecandidate ANT ligand. Those having familiarity with the art willappreciate that variations and modifications may be made to ANT-bindingassays such as those illustrated above and described in the Examples fordeterming IC₅₀ values of candidate ANT ligands, and which are notintended to be limiting.

Activity of each ANT ligand is reported as a “K_(i)” value calculated bythe following equation: $K_{i} = \frac{{IC}_{50}}{1 + {L/K_{D}}}$where L=radioligand and K_(D)=affinity of radioligand for receptor(Cheng and Prusoff, Biochem. Pharmacol. 22:3099, 1973). ANT ligands ofthis invention have a K_(i) of 100 μM or less. In a preferred embodimentof this invention, the ANT ligands have a K_(i) of less than 10 μM, andmore preferably less than 1 μM. To this end, ANT ligands of thisinvention having a K_(i) of less than 100 μM include compound 5 (Example7), compound 6 (Example 8), and compounds 22, 23, 24, 26, 29, 33, 35,37, and 38 (Example 11). Preferred ANT ligands having a K_(i) of lessthan 10 μM include compounds 6, 22, 23, 24, 29, 33, 35, and 38, and morepreferred ANT ligands having a K_(i) of less than 1 μM include compounds6, 24, 33, and 38, as well as ATR.Assays

It is another aspect of the invention to provide compositions andmethods for the determination of the presence of ANT polypeptides andfor the identification of agents that bind to, or that interact with,ANT polypeptides. Such compositions and methods will be useful fordiagnostic and prognostic purposes, for example in the determination ofthe existence of altered mitochondrial function which, as describedabove, may accompany both normal and disease states. These compositionsand methods will also be useful for the identification of agents thatalter or regulate mitochondrial function based on ANT roles inmitochondrial activities, for example by way of illustration and notlimitation, maintenance of mitochondrial membrane potential, ATPbiosynthesis, induction of apoptosis, MPT and other mitochondrialfunction. In certain preferred embodiments these compositions andmethods are useful as high throughput screening assays.

In certain aspects the invention provides a method for determining thepresence of an ANT polypeptide in a biological sample, comprisingcontacting a sample suspected of containing an ANT polypeptide with anANT ligand under conditions and for a time sufficieint to allow bindingof the ANT ligand to an ANT polypeptide, and detecting such binding.“ANT ligands” according to these aspects of the invention may includeany novel ANT ligands as provided herein. The use of human ANT1, ANT2and ANT3 according to these methods represent particularly preferredembodiments. Other preferred embodiments include the use of any ANTpolypeptide or ANT fusion protein as provided herein. Accordingly, theinstant method for determining the presence of ANT polypeptide in asample will be useful for monitoring expression of ANT encodingconstructs provided herein. In some preferred embodiments an ANT fusionprotein is used that is a GST fusion protein, and in other preferredembodiments the ANT fusion protein is a His-tagged fusion protein. Asprovided herein, the biological sample may be a cell, a mitochondrion,submitochondrial particles, a cell membrane (including any cellularmembrane as described herein), a cell extract, cell conditioned medium,a tissue homogenate or an isolated ANT.

In other aspects, the invention provides a method for identifying anagent that binds to an ANT polypeptide, comprising contacting acandidate agent with a host cell expressing at least one recombinant ANTpolypeptide under conditions and for a time sufficient to permit bindingof the agent to the ANT polypeptide and detecting such binding. Invarious preferred embodiments the host cell may be a prokaryotic cell ora eukaryotic cell. In certain other preferred embodiments the host cellmay lack at least one isoform of an endogenous ANT, for example, due toa mutation in one or more endogenous ANT encoding genes. In certainother embodiments host cell expression of at least one gene encoding anendogenous ANT isoform is substantially impaired, for example, throughthe use of ANT nucleic acid-specific ribozyme or antisense constructs asprovided herein, or through the use of ρ⁰ cells, as also providedherein. According to other embodiments of this aspect of the invention,it may be preferred to use intact cells or, alternatively, to usepermeabilized cells. Those having ordinary skill in the art are familiarwith methods for permeabilizing cells, for example by way ofillustration and not limitation, through the use of surfactants,detergents, phospholipids, phospholipid binding proteins, enzymes, viralmembrane fusion proteins and the like; through the use of osmoticallyactive agents; by using chemical crosslinking agents; by physicochemicalmethods including electroporation and the like, or by otherpermeabilizing methodologies.

In other aspects, the invention provides a method for identifying anagent that binds to an ANT polypeptide comprising contacting a candidateagent with a biological sample containing at least one recombinant ANTpolypeptide under conditions and for a time sufficient to permit bindingof the agent to the ANT polypeptide, and detecting such binding. The useof human ANT1, ANT2 and ANT3 according to these methods representparticularly preferred embodiments. Other preferred embodiments includethe use of any ANT polypeptide or ANT fusion protein as provided herein.In some preferred embodiments an ANT fusion protein is used that is aGST fusion protein, and in other preferred embodiments the ANT fusionprotein is a His-tagged fusion protein. As provided herein, thebiological sample may be a cell, a mitochondrion, submitochondrialparticles, a cell membrane (including any cellular membrane as describedherein), a cell extract, cell conditioned medium, a recombinant viralparticle, a tissue homogenate or an isolated ANT. Detection of bindingmay be by any of a variety of methods and will depend on the nature ofthe candidate agent being screened. For example, certain candidateagents are inherently detectable as a consequence of theirphysicochemical properties, such as will be apparent to those skilled inthe art and including spectrophotometric, colorimetric, fluorimetric,solubility, hydrophobic, hydrophilic, electrostatic charge, molecularmass or other physicochemical properties. As another example, certaincandidate agents may be radioactively labeled with a readily detectableradionuclide, as is well known in the art. Certain candidate agents mayalso be directly or indirectly detectable by ANT protein affinitymethodologies, for example by their ability to interfere with binding ofan ANT-specific antibody to an ANT; or by their being removable from anassay solution using a protein affinity reagent that binds to a fusionpolypeptide present as a portion of an ANT fusion protein. A candidateagent bound to an ANT polypeptide may be detected by any method knownfor the detection, identification or characterization of relevantmolecules, including spectrophotometric, mass spectrometric,chromatographic, electrophoretic, calorimetric or any other suitableanalytical technique.

In another aspect the invention provides a method for identifying anagent that interacts with an ANT polypeptide comprising contacting abiological sample containing recombinant ANT with a detectable ANTligand (or a known detectable molecule capable of binding to ANT) in thepresence of a candidate agent, and comparing binding of the detectableANT ligand (or known detectable ANT binding molecule) to recombinant ANTin the absence of the agent to binding of the detectable ANT ligand (orknown detectable ANT binding molecule) to recombinant ANT in thepresence of the agent, and therefrom identifying an agent that interactswith an ANT polypeptide. It will be appreciated that in certainpreferred embodiments this aspect provides competitive binding assayswherein novel ANT ligands as provided hereinabove are useful. However,this aspect of the invention need not be so limited and may be modifiedto employ known detectable ANT binding molecules, in which case itshould be pointed out that the selection of biological sample and/or ofrecombinant ANT as provided by the present invention offer unexpectedadvantages heretofore unknown in the art. Examples of known detectableANT-binding molecules include suitably labeled ATP, ADP, ATR, CATR,palmitoyl-CoA, bongkrekic acid, thyroxin, eosin Y and erythrosin B orother ANT-binding molecules known in the art. (See, e.g., Block et al.,1986 Meths. Enzymol. 125:658.) The use of human ANT1, ANT2 and ANT3according to these methods represent particularly preferred embodiments.Other preferred embodiments include the use of any ANT polypeptide orANT fusion protein as provided herein. In some preferred embodiments anANT fusion protein is used that is a GST fusion protein, and in otherpreferred embodiments the ANT fusion protein is a His-tagged fusionprotein. As provided herein, the biological sample may be a cell, amitochondrion, submitochondrial particles, a cell membrane (includingany cellular membrane as described herein), a cell extract, cellconditioned medium, a recombinant viral particle, a tissue homogenate oran isolated ANT.

The ANT ligands compounds are preferably part of a pharmaceuticalcomposition when used in the methods of the present invention. Thepharmaceutical composition will include at least one of apharmaceutically acceptable carrier, diluent or excipient, in additionto one or more ANT ligands and, optionally, other components.

“Pharmaceutically acceptable carriers” for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compounds of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions that contain one or more ANTsubstrates/ligands compounds may be in any form which allows for thecomposition to be administered to a patient. For example, thecomposition may be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal, intracavernous, intrameatal, intraurethral injection orinfusion techniques. The pharmaceutical composition is formulated so asto allow the active ingredients contained therein to be bioavailableupon administration of the composition to a patient. Compositions thatwill be administered to a patient take the form of one or more dosageunits, where for example, a tablet may be a single dosage unit, and acontainer of one or more compounds of the invention in aerosol form mayhold a plurality of dosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to one or more ANT substrates/ligands compounds, one or more ofa sweetening agent, preservatives, dye/colorant and flavor enhancer. Ina composition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oraladministration should contain an amount of ANT substrates/ligandscompound such that a suitable dosage will be obtained. Typically, thisamount is at least 0.01 wt % of an ANT substrates/ligands compound inthe composition. When intended for oral administration, this amount maybe varied to be between 0.1 and about 70% of the weight of thecomposition. Preferred oral compositions contain between about 4% andabout 50% of ANT substrates/ligands compound(s). Preferred compositionsand preparations are prepared so that a parenteral dosage unit containsbetween 0.01 to 1% by weight of active compound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the ANT substrates/ligands compound of from about 0.1to about 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

In the methods of the invention, the ANT substrates/ligands compound(s)may be administered through use of insert(s), bead(s), timed-releaseformulation(s), patch(es) or fast-release formulation(s).

It will be evident to those of ordinary skill in the art that theoptimal dosage of the ANT substrates/ligands compound(s) may depend onthe weight and physical condition of the patient; on the severity andlongevity of the physical condition being treated; on the particularform of the active ingredient, the manner of administration and thecomposition employed. It is to be understood that use of an ANTsubstrates/ligands compound in a chemotherapy can involve such acompound being bound to an agent, for example, a monoclonal orpolyclonal antibody, a protein or a liposome, which assist the deliveryof said compound.

EXAMPLES

The following Examples are offered by way of illustration and not by wayof limitation.

Example 1 Cloning and Expression of His-Tagged Human ANT Proteins inBacteria

A. PCR Amplification of ANT cDNAs

Total cellular RNA prepared from whole human brain was obtained from acommercial source (Clontech, Palo Alto, Calif.). The RNA was purified bytreatment with RNase-free DNase I (Roche Molecular Biochemicals,formerly Boehringer Mannheim Biochemicals, Indianapolis, Ind.) using 1ul of DNase I (10 u/ul) in a buffer containing 40 mM Trsi-HCl, pH 7.0, 6mM magnesium chloride and 2 mM calcium chloride for 30 minutes at 37° C.This treatment was followed by two phenol/chloroform extractions, onechloroform extraction and an ethanol precipitation in the presence ofsodium acetate. The RNA pellet was collected by centrifugation, washedwith 70% ethanol, air dried, and resuspended in RNase-free sterilewater. The RNA was reverse transcribed to generate cDNA using RNaseH-deficient Reverse Transcriptase (SUPERSCRIPT™; Life Technologies,Rockville, Md.).

ANT cDNAs were amplified by polymerase chain reactions (PCR) in athermal cycler using the following primers, AMPLITAQ™ DNA Polymerase(Perkin-Elmer), and reagents and buffers supplied in a GENEAMP™ PCRReagent Kit (Perkin-Elmer), according to the manufacturer'sinstructions. In the following representations of the PCR primers,underlined nucleotides indicate sequences complementary to the 5′-endsand 3′-ends of the ANT cDNAs and double-underlined nucleotides indicaterecognition sequences for the restriction enzymes XhoI (recognitionsequence: 5′-CTCGAG) and Asp718 (recognition sequence: 5′-GGTACC).

For human ANT1 (huANT1; SEQ ID NO:1), the following primers were used:Forward (sense): 5′-TTATATCTCGAGTATGGGTGATCACGCTTGGAGCTTCCTAAAG SEQ IDNO:4 and Reverse (antisense): 5′-TATATAGGTACCTTAGACATATTTTTTGATCTCATCATACAAC. SEQ ID NO:5 For human ANT2 (huANT2; SEQID NO:2), the following primers were used: Forward (sense):5′-TTATATCTCGAGTATGACAGATGCCGCTGTGTCCTTCGCCAAG SEQ ID NO:6 and Reverse(antisense): 5′-TATATAGGTACC TTATGTGTACTTCTTGATTTCATCATACAAG. SEQ IDNO:7 For human ANT3 (huANT3; SEQ ID NO:3), the following primers wereused: Forward (sense): 5′-TTATATCTCGAGTATGACGGAACAGGCCATCTCCTTCGCCAAASEQ ID NO:8 and Reverse (antisense): 5′-TATATAGGTACCTTAGATCACCTTCTTGAGCTCGTCGTACAGG. SEQ ID NO:9B. Generation of ANT Expression Constructs

PCR products were digested with the restriction endonucleases XhoI andAsp718 (both enzymes from Roche Molecular Biochemicals) according to themanufacturer's recommendations using manufacturer-supplied reactionbuffers. Restricted DNAs were purified by horizontal agarose gelelectrophoresis and band extraction using the UltraClean GelSpin kit (MoBio Laboratories, Inc., Solana Beach, Calif.).

The expression vector pBAD/His (“B” derivative; Invitrogen, Carlsbad,Calif.) was used. This vector contains the following elements operablylinked in a 5′ to 3′ orientation: the inducible, but tightlyregulatable, araBAD promoter; optimized E. coli translation initiationsignals; an amino terminal polyhistidine(6×His)-encoding sequence (alsoreferred to as a “His-Tag”); an XPRESS™ epitope-encoding sequence; anenterokinase cleavage site which can be used to remove the precedingN-terminal amino acids following protein purification, if so desired; amultiple cloning site; and an in-frame termination codon.

Plasmid pBAD/His DNA was prepared by digestion with the restrictionendonucleases XhoI and Asp718 according to the manufacturer'sinstructions and subjected to horizontal agarose gel electrophoresis andband extraction using the UltraClean GelSpin kit (Mo Bio Laboratories).Restricted ANT cDNAs were ligated into the linearized plasmid withrestricted expression vector DNA using T4 DNA ligase (New EnglandBiolabs, Beverly, Mass.) using the manufacturer's reaction buffer andfollowing the manufacturer's instructions. Competent recA1 hsdR endA1E.coli cells (strain TOP10F′; Invitrogen, Catalog #C3030-03) weretransformed with ligation mixtures containing the prokaryotic vectorconstruct according to the manufacturer's instructions. Single colonieswere selected and grown in 3-5 ml of LB broth (Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)containing 50 μg/ml ampicillin (Roche Molecular Biochemicals). PlasmidDNA was isolated from the bacterial cultures using the WIZARD™ PlusSeries 9600 Miniprep Reagents System (Promega, Madison, Wis.).

The recombinant huANT nucleotide sequences present in the expressionconstructs were determined and their authenticity confirmed relative tothe published ANT sequences (FIG. 1; See Neckelmann et al., Proc. Nat'l.Acad. Sci. U.S.A. 84:7580-7584 (1987) for huANT1; Battini et al., J.Biol. Chem. 262:4355-4359 (1987) for huANT2, and Cozens et al., J. Mol.Biol. 206:261-280 (1989) for huANT3.) by DNA sequencing using the PRISM™Ready BIG DYE™ Terminator Cycle Sequencing Kit (The Perkin-Elmer Corp.,Norwalk, Conn.) and the following sequencing primers5′-TATGCCATAGCATTTTTATCC (SEQ ID NO:10) and 5′-CGCCAAAACAGCCAAGCT (SEQID NO:11). For each human ANT sequence, both primers are located insidethe vector sequence adjacent to the DNA insertion. Sequence data wasanalyzed using the SEQUENCE NAVIGATOR™ analysis software package(Perkin-Elmer). This huANT3 expression construct was named pMK3A-huANT3.

The expression plasmids encoding His-tagged human ANT1, ANT2 and ANT3are referred to herein as follows: For human ANT1, “pMK1 (His-taggedhuANT1)” or “pMK1”; for human ANT2, “pMK2 (His-tagged huANT2)” or“pMK2”; for human ANT3 “pMK3A (His-tagged hu ANT3” or “pMK3A”; for humanANT3 from which extraneous linker N-terminal amino acids are deleted asdetailed below, “pMK3B (His-tagged hu ANT3, shortened epiotpe linker)”or “pMK3B”. Plasmids pMK1, pMK2 and pMK3A have been deposited at theAmerican Type Culture Collection (ATCC; Manassas, Va.) on Nov. 3, 1998,and given the accession numbers ATCC 98969, ATCC 98970 and ATCC 98971,respectively.

The expression constructs comprising nucleotide sequences encoding humanANT1 (pMK1-huANT1) and human ANT2 (pMK2-huANT2) were restriction mappedto confirm their structures. The nucleotide sequences of plasmidspMK1-huANT1 and pMK2-huANT2 are determined using the methods and primers(SEQ ID NOS:10 and 11) described above.

Treatment of the recombinant huANT3 protein expressed from pMK3A-huANT3with enterokinase liberates the His-Tag/XPRESS™ epitope polypeptide fromthe huANT3 protein; however, the resultant huANT3 protein comprisesseveral extraneous N-terminal amino acids (i.e., Pro-Ser-Ser-Ser-Met,where “Met” indicates the amino acid encoded by the translationinitiation codon of huANT3). Although the extraneous amino acidsprobably have little or no effect on the recombinant huANT3 protein, aderivative expression construct in which the nucleotide sequenceencoding the extraneous amino acids are deleted was prepared in thefollowing manner.

The QUIK-CHANGE™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.) essentially according to the manufacturer's instructions.Briefly, a reaction mixture comprising purified pMK3A-huANT3 DNA, themutagenic oligonucleotide primers5′-GGAGATGGCCTGTTCCGTCATCTTATCGTCATCGTCGTACAGATC (SEQ ID NO:12; theunderlined sequence is the reverse complement of the 5′ end of thehuANT3 reading frame), and5′-GATCTGTACGACGATGACGATAAGATGACGGAACAGGCCATCTCC(SEQ ID NO:13; theunderlined sequence corresponds to the 5′ end of the huANT3 readingframe), Pfu DNA polymerase and dNTPs in manufacturer-supplied reactionbuffer was prepared. The mutagenic oligonucleotide primers were presentin excess and cycles of DNA synthesis was carried out in a thermalcycler according to the manufacturer's protocol. The reaction productswere treated with the restriction enzyme DpnI, which cleaves methylatedand hemi-methylated DNAs but leaves unmethylated DNA (i.e., annealedproducts of the reaction) intact, and used to transform EPICUREAN COLI™XL-1-Blue E. coli cells (Stratagene). Plasmid DNA was prepared fromtwelve randomly selected transformants and the nucleotide sequence ofthe region containing the multiple cloning site cassette was determinedaccording to the methods described above. Of the twelve plasmids, onlyone retained the original sequence found in pMK-huANT3, and threecontained undesired point mutations. One of the eight “correct” plasmidswas chosen and named pMK3B-huANT3.

C. Expression of His-Tagged huANT3

Cultures of E. coli cells containing pMK3A-huANT3 were grown in LB mediacontaining 50 ug/ml ampicillin to mid-log phase (OD₆₀₀˜0.5) and inducedfor 3-4 hours with increasing doses of arabinose (i.e., 0.00002%,0.0002%, 0.002%, 0.02%, and 0.2%). One ml of each culture wascentrifuged at 5,000×g for 10 minutes at 4° C. to pellet the cells. Cellpellets were resuspended, and the cells were lysed, by adding 100 ul ofPhosphate Buffered Saline (PBS; pH 7.4) containing 1% cholate, 1%n-dodecyl maltoside, and 0.1% 2-mercaptoethanol (in the preceding text,and throughout the specification, unless specified otherwise, allchemicals are from Sigma, St. Louis, Mo.). Total protein content in thelysates was determined using the BCA (bicinchoninic acid; Smith et al.,1985, Anal. Biochem. 150:76-85) Protein Assay kit (Pierce Chemical Co.,Rockford, Ill.). Ten μg of total protein were loaded per lane onto anSDS polyacrylamide gel, electrophoresed and transferred to anitrocellulose membrane (HYBOND™ ECL Nitrocellulose Membrane, AmershamPharmacia Biotech, formerely Amersham Life Sciences, Piscataway, N.J.).Human ANT3 fusion proteins were detected in a western blot usingANT1-XPRESS™ Antibody (Invitrogen) and horseradish peroxidase-conjugatedanti-mouse secondary antibody (Amersham Pharmacia Biotech) according tothe manufacturers' instructions.

The results are shown in FIG. 3. From left to right in the figure, thefollowing samples are shown: lanes “M”, molecular weight markers; lane“0”, untransformed E. coli cells; lane “o/n”, E. coli comprisingpMK3A-huANT3 grown overnight without induction; lane “1”-“5”, E. colicomprising pMK3A-huANT3 grown induced with increasing doses of arabinose(0.00002%, 0.0002%, 0.002%, 0.02% and 0.2%, respectively). As expected,untransformed (lane 0) and uninduced (lane o/n) E. coli showed noXPRESS™-huANT3 material. However, expression of recombinant ANT3 fusionprotein with a molecular weight of 36.6 kD was observed in lanes 3 and 4(0.002% and 0.02% arabinose, respectively). No XPRESS™-huANT3 materialwas detected in lanes 1 and 2 (0.00002% and 0.0002% arabinose,respectively) indicating that the degree of induction was insufficientunder these conditions.

Cells that were grown in the presence of the highest concentration ofarabinose (0.2%, lane 5) began to lyse and died before the time ofharvest; consequently, no recombinant protein was detected. Thisindicated that very high expression of recombinant huANT in E. colicaused cell death, as is sometimes the case during overexpression ofheterologous proteins in bacteria.

D. Recombinant huANT3 Localizes to the Bacterial Membrane

In order to locate the expressed human ANT 3 within E. coli cells, cellswere grown in culture and induced with arabinose as described above, andthen fractionated into different compartments (e.g., membranes,inclusion bodies and cytosol). Bacteria were pelleted by centrifugationat 5,000×g for 10 minutes at 4° C. The cell pellets were resuspended in1/10 volume of cell buffer A (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100ug/ml lysozyme, and 0.1% Triton X-100) and incubated for 15 minutes at30° C. in an orbital shaker. The cell mixture was sonicated for 2minutes and membranes were pelleted by centrifugation at 12,000×g for 15minutes at 4° C. The supernatant, representing the cytosol, was removedfor analysis (FIG. 4, lane 4), as was a portion of the pellet containingmembranes and inclusion bodies (FIG. 4, lane 3). The remaining portionof the pellet was washed twice with cell buffer B (10 mM Tris-HCl, pH7.0, 0.1 mM EDTA, and 1 mM DTT) and centrifuged at 12,000×g for 15minutes at 4° C. The pellet was resuspended in cell buffer C (20 mMTris-HCl, pH 8.0, 100 mM sodium chloride, and 6 M guanidiniumhyrochloride) and incubated for 1 hour at room temperature. The solutionwas then centrifuged at 12,000×g for 15 minutes at 4° C. The supernatant(containing solubilized inclusion bodies; lane 1, FIG. 4) and the pellet(containing insoluble inclusion bodies; lane 2, FIG. 4) were analyzed byWestern blotting as described above.

The results are shown in FIG. 4. Recombinant huANT 3 (molecular weight36.6 kD) was detected in lanes 2, 3, and 4, as well as the positivecontrol lane (+) (total cell lysate previously tested for presence ofANT3 protein by Western immunoblot analysis, as described above). Thegreatest amount of recombinant huATN3 was detected in lane 3, whichrepresents the membrane fraction. This indicates that the majority ofthe huANT3 fusion protein integrated into the E. coli cellular membrane.Smaller protein signals were visible in lanes 2 and 4, representing theinsoluble inclusion body fraction which might have contained somemembranes with integrated ANT 3, and the cytosolic fraction whereprotein synthesis takes place. No protein was detectable in the solubleinclusion body fraction in lane 1, indicating that controlled expressionof ANT3 in the bacteria did not result in the formation of inclusionbodies, which is an undesirable consequence of over-expression of someheterologous proteins in bacteria.

E. Purification of ANT Proteins

ANT proteins, and ANT fusion proteins, produced by the expressionsystems described herein have been purified using a variety of methods.The purification of ANT proteins, particularly human ANT proteins, isdescribed in this Example.

Regardless of which of following protein purification methods is used,or others that can be derived from the present disclosure, it isimportant to add sufficient amounts of DNase and RNase to eliminate theviscosity associated with some bacterial lysates (typically 10 μg/mL ofeach enzyme; both from Roche Biochemicals) when the bacterial cells arelysed (or immediately thereafter). An alternative or additional means bywhich viscosity has been minimized and ANT solubility has been optimizedis vigorous sonication, as opposed to standard sonication, of thelysates. The term “vigorous sonication” refers to, for example,sonication with a Branson Sonifier (Model 450) 2×(30 seconds each time)at 50% duty cycle and 80% output using a tapered, flat-tipped probe (asopposed to sonication with a cup and horn apparatus). Although eithertype of sonication will suffice, better yields have typically beenobserved when vigorous sonication has been used.

Furthermore, in various ANT purification methods that have been used, itwas often desirable to make the lysate at least 1% Triton-X, in order tosolubilize the maximum possible amount of ANT protein, after whichinsoluble material is removed by a high-speed (i.e., about 100,000 g)spin. Typically, protease inhibitors such as, for example, pepstatin,leupeptin, phenylmethylsulfonyl fluoride (PMSF) and/or aprotinin (allfrom Sigma) have been present at effective levels (typically 10 μg/mL)during the preparation. Depending on the particular ANT protein or ANTfusion protein being isolated, all four protease inhibitors or anyeffective combination thereof are used. For example, in preparations ofGST-huANT3 fusion proteins, best results were seen when all fourprotease inhibitors were used, although acceptable results have beenobtained when only leupeptin and pepstatin were used.

One method incorporates novel methods with several techniques previouslyused only for purifying ANT proteins from non-human mammals, i.e.,bovine cardiac tissue and rats (Aquila et al., 1982, Hoppe-Seyler's Z.Physiol. Chem. 363:345-349; and Sterling, 1986, Endocrinology119:292-295). In brief, bacterial cells expressing a GST-ANT3 fusionprotein were lysed by lysozyme treatment, and ¹⁴C-palmityl-CoA (Sigma)was added at a concentration of 50 nmol per gram of E. coli. Because itassociates with ANT proteins, ¹⁴C-palmityl-CoA acts as a radiolabeledtracer that can be used to follow the ANT protein in subsequentpurification steps. The lysates were then sonicated and made 6% TritonX-100 (Sigma) and incubated at 4° C. for 1 hr to solubilize material. Ahigh-speed spin was used to remove insoluble material, and the resultingsolute was applied either (1) for small scale preparations, tohydroxyapatite beads (Bio-Rad Laboratories, Hercules, Calif.), or (2) inthe case of larger preparations (i.e., ≧1 liter of bacterial culture),to a hydroxyapatite column (Bio-Rad) essentially according to themanufacturer's instructions. Unlike other intramembrane mitochondrialproteins, ANT has a low affinity for hydroxyapatite (Klingenberg et al.,1978, Biochim. et Biophys. Acta 503:193-210). The hydroxyapatite columnwas eluted with Column Buffer A (10 mM MOPS, pH 7.2, 100 Mm NaCl, 9.5%Triton X100) and washed with Column Buffer B (10 mM MOPS, pH 7.2, 100 mMNaCl, 400 mM sodium phosphate). Non-recombinant ANT proteins fromnon-human species are eluted in the void volume with Column Buffer A,and the GST-huANT3 fusion protein was expected to be present in the voidvolume as well; Column Buffer B was used to wash the column in the eventthat GST-huANT3 fusion protein behaves differently. Samples werecollected in such a manner as to have a final concentration of 30 of mMoctyl glucoside (Calbiochem), a nonionic detergent that helps solubulizeANT proteins with minimal effect on activity (Sterling, 1986,Endrocrinol. 119: 292-295). The bead-extracted supernatant or columneluent was collected, and Triton X-100 was removed therefrom using theEXTRACTI-GEL™ affinity matrix (Pierce) essentially according to themanufacturer's instructions (see also Berman et al., 1985, Biochemistry24:7140-7147).

Varying amounts of GST-huANT3 prepared in the above manner were subjectto PAGE and the gel was stained using a colloidal blue protein stain(Novex, San Diego, Calif.). The stained gel displayed a single bandhaving a molecular weight corresponding to that predicted for the fusionprotein. Based on the intensity of bands from samples of varyingvolumes, and the known volume of the preparation and minimal sensitivityof the stain, the yield from 100 mL of bacterial culture was estimatedto be about 50 ug. In one of the lanes of the gel, approximately 500 ngof protein was loaded, and no contaminating bands were detected; thisindicates that the GST-huANT3 protein was from at least about 90% pureto at least about 95% pure.

GST-huANT3 fusion proteins (see preceding Examples) have been purifiedby this method, and other ANT fusion proteins, including His-taggedhuANT3 and other His-tagged ANT proteins, are purified in like fashion.Purified huANT fusion proteins are used to produce purified human ANTproteins as follows.

GST-huANT fusion proteins are further purified via glutathione-agarosebeads (Sigma) essentially according to the manufacturer's instructions.In brief, a solution comprising GST-huANT fusion proteins is contactedwith glutathione-agarose beads, and the beads are washed to releaseundesirable contaminants. Next, the [bead:GST-huANT] complexes aretreated with an appropriate enzyme, i.e., one that separates the huANTpolypeptide from the remainder of the fusion protein. In the case of theGST-huANT3 fusion protein described herein (i.e., that encoded bypMK3C), thrombin (Sigma) cleaves the fusion protein in such a manner soas to produce two polypeptides: a first polypeptide corresponding to theGST moiety, and a second polypeptide which corresponds to human ANT3with an additional six amino acids (i.e., Gly-Ser-Pro-Gly-Ile-Leu)present at its N-terminus.

His-tagged huANT fusion proteins are further purified via Nickel-coatedresins (such as, e.g., PROBOND™ Ni²⁺ charged agarose resin; Invitrogen)essentially according to the manufacturer's instructions. In brief, asolution comprising His-tagged huANT fusion proteins is contacted withthe Nickel-coated resin, and the resin is washed to release undesirablecontaminants. Next, the [resin:His-tagged huANT] complexes are treatedwith an appropriate enzyme, i.e., one that separates the huANTpolypeptide from the remainder of the fusion protein. In the case of theHis-tagged huANT3 fusion proteins described herein, enterokinase (Sigma,or EKMAX™ from Invitrogen may be used) cleaves the fusion protein insuch a manner so as to produce two polypeptides: a first polypeptidecomprising the His-tag and XPRESS™ epitope moieties, and a secondpolypeptide which corresponds to human ANT3. If the expression constructused is pMK3A, the resultant purified human ANT3 protein has anadditional four amino acids (i.e., Pro-Ser-Ser-Ser) at its N-terminus.If pMK3B is the expression construct present in the cells from whichHis-tagged huANT3 is isolated, the resultant purified human ANT3 proteinhas the sequence of native huANT3, i.e., SEQ ID NO:3.

In both of the preceding purification steps, an ANT fusion protein boundto a solid support is treated with an enzyme (i.e., thrombin orenterokinase) that liberates an ANT protein from the remainder of thefusion protein, which remains bound to the solid support. ANT protein isreleased into the liquid phase which is then collected to generate asolution comprising the ANT protein and some amount of the liberatingenzyme. The amount of liberating enzyme needed is minimal because thetreatment is catalytic in nature; nevertheless, some enzyme remains inthe preparation. If desired, enzyme molecules may be removed from thepreparation using any of a variety of means known in the art. Forexample, an enzyme may be removed from a solution by contacting thesolution with a resin conjugated to a ligand having a high affinity forthe enzyme. In the case of enterokinases, one such resin is the EK-AWAY™resin (Invitrogen) which comprises the soybean trypsin inhibitor havinga high affinity for enterokinases. Methods of treating GST fusionproteins with thrombin and purifying the desired recombinant proteinhave been described previously (see, for example, Smith and Corcoran,Unit 16.7 in Chapter 16 in Short Protocols in Molecular Biology 2^(nd)Ed., Ausubel et al., eds, John Wiley & Sons, New York, N.Y., 1992, pages16-28 to 16-31. In general, however, any suitable means for separatingthe liberating enzyme from any given ANT protein may be used.

F. Growth Inhibition

As noted in the above discussion of the results presented in FIG. 3,very high expression of recombinant huANT3 in E. coli caused cell death.Such a result is sometimes observed during over-expression ofheterologous proteins in bacteria. Although not wishing to be bound byany particular theory, because the recombinant huANT3 protein localizedto the bacterial membrane, and because ANT3 functions as an ATP/ADPexchanger in the inner mitochondrial membrane and under appropriateconditions may exhibit pore properties suggestive of a role in membranepermeability, one possible explanation for the observed cell death wouldbe an inappropriate enhancement of the permeability of the bacterialmembrane. If this in fact the case, inhibitors of mitochondrial ANTmight prevent the death of E. coli overexpressing huANT3. As notedabove, under certain conditions atractyloside or bongkrekic acid mayexhibit inhibition of ANT activity, such that either of theseinhibitors, other known ANT-active agents and potentially other ANTligands as provided herein may be employed in the instant Exampledescribed using bongkrekic acid.

In order to test this hypothesis, the following experiments are carriedout. E. coli harboring pMK3A-huANT3 are grown with no arabinose or with0.2% or more arabinose, the latter concentration having been previouslyshown to induce toxic levels of huANT3, and various concentrations (0,5, 20, 50 and 200 μM) of bongkrekic acid (Biomol Research Laboratories,Inc., Plymouth Meeting, Mass.), an inhibitor of ANT (Henderson andLardy, 1970, J. Biol. Chem. 245:1319-1326) that binds to ANT (see, e.g.,Vignais et al., 1976, Biochim. Biophys. 440:688-696). The ability ofbongkrekic acid to prevent the lysis of E. coli overexpressing huANT3,or any other ANT protein for that matter, indicates that the toxiceffect of such overexpression is due to an activity associated withnormally functioning ANT.

ANT proteins produced by this expression system, and others describedherein, are also purified using known methods for purifying ANT proteinsfrom humans and other mammals. See for example, Klingenberg et al., 1978Biochim. Biophys. Acta 503:193-210; Aquila et al., 1982 Hoppe-Seyler'sZ. Physiol Chem. 363:345-349; and Sterling, 1986 Endocrinol.119:292-295.

The bacterial toxicity of extreme overexpression of ANT in this systemcan be used to screen and identify novel inhibitors of ANT, as suchcompounds will be expected to also prevent lysis of E. colioverexpressing ANT proteins. In order to achieve a greater degree ofspecificity for the ANT protein produced from an expression vector, theyeast expression system for ANT proteins (see Example 4, infra) is usedin a mutant yeast strain that is resistant to bongkrekic acid (Lauquinet al., 1975 FEBS Letters 35:198-200).

Example 2 Expression of GST-HuANT3 Fusion Proteins

A. Generation of GST-huANT3 Expression Constructs

Human ANT3 cDNA was amplified from pMK3A-huANT3 by PCR as in Example 1but using the following primers. In the following representations of PCRprimers, underlined nucleotides indicate sequences complementary to the5′-ends and 3′-ends of the ANT cDNAs and double-underlined nucleotidesindicate recognition sequences for the restriction enzymes XhoI(recognition sequence: 5′-CTCGAG) or EcoRI (recognition sequence:5′-GAATTC).

The primers used for PCR amplification were: Forward (sense): SEQ IDNO:14 5′-CCCGGGGAATTCTGATGACGGAACAGGCCATCTCC and Reverse (antisense):SEQ ID NO:15 5′-CCCGGGCTCGAG TTAGAGTCACCTTCTTGAGCTC

The expression vector pGEX-4T-2 (Amersham Pharmacia Biotech) was used togenerate huANT3 fusion proteins comprising an enzymatic polypeptide andan ANT polypeptide. This vector comprises a lacI^(q) (repressor) gene atac promoter operably linked to a glutathione S-transferase (GST) genefrom Schistosoma japonicum. (Smith et al., 1988, Gene 67:31-40), thecoding sequence of which has been modified to comprise a thrombincleavage site-encoding nucleotide sequence immediately 5′ from amultiple cloning site. GST fusion proteins can be detected by Westernblots with anti-GST or by using a colorimetric assay; the latter assayutilizes glutathione and 1-chloro-2-4-dinitrobenzene (CDNB) assubstrates for GST and yields a yellow product detectable at 340 nm(Habig et al., 1974, J. Biol. Chem. 249:7130-7139). GST fusion proteinsproduced from expression constructs derived from this expression vectorcan be purified by, e.g., glutathione affinity chromatography, and thedesired polypeptide released from the fusion product by thrombin. Thus,this expression vector provides for the rapid purification of fusionproteins, and release of proteins with relatively few extraneousN-terminal amino acids, although the resulting recombinantly producedprotein contains two additional amino acids at the amino terminus(Gly-Ser). The tac promoter may be induced by the addition to culturedcells of, e.g., 1-5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG;Fluka, Milwaukee, Wis.) and provides for high-level expression.

Plasmid pGEX-4T-2 was prepared by digestion with the restrictionendonucleases EcoRI and Asp718 according to the manufacturer'sinstructions and subjected to horizontal agarose gel electrophoresis andband extraction using the UltraClean GelSpin kit (Mo Bio Laboratories).Restricted ANT cDNAs were ligated with the restricted expression vectorDNA as described in the preceding Example. Single colonies were selectedfor grown in 3-5 ml of LB broth containing 50 ug/ml ampicillin (RocheMolecular Biochemicals), and plasmid DNA was isolated from the bacterialcultures using the WIZARD™ Plus Series 9600 Miniprep Reagents System(Promega). To confirm their authenticity, the recombinant huANTnucleotide sequences present in the pGEX deriavtive plasmid weredetermined as described in the preceding Example using the previouslydescribed oligonucleotide primers and 5′ and 3′ PGEX Sequencing Primers(Amersham Pharmacia Biotech).

The resultant GST-huANT3 expression construct was named pMK3C-GST-huANT3(also referred to herein as pMK3C). Plasmid pMK3C has been deposited atthe American Type Culture Collection (ATCC; Manassas, Va.) on Nov. 3,1998, and given the accession number ATCC 98973. Thrombin treatedrecombinant huANT3 protein produced from the pMK3C-GST-huANT3 expressionconstruct includes several extraneous N-terminal amino acids, i.e.,Gly-Ser-Pro-Gly-Ile-Leu-Met, where “Met” indicates the amino acidencoded by the translation initiation codon of huANT3. There is,however, no evidence that the extraneous six amino terminal amino acidshave any effect on the resultant recombinant huANT3 protein.

In order to confirm expression of the GST-huANT3 fusion protein, thefollowing experiments were carried out. Eight independently isolatedpMK3C-GST-huANT3 transformants and one control (vector-transformed)isolate were grown overnight in LB-ampicillin and then diluted 1:20 in 2ml of fresh media. After 3 hours of growth at 37° C., IPTG was added toa final concentration of 0.1 mM. Cell growth was continued for 2 hours,after which 1.5 of cells were tranferred to microfuge tubes, pelleted,resuspended in 300 uL of cold PBS containing 1% Triton X-100, andsonicated twice for 8 seconds. The sonicates were spun for 5 min. at 4°C., the supernatant was transferred to fresh microfuge tubes and 50 uLof glutathione-agarose beads (Sigma) were added to produce a 50% slurry.After a 5 min. incubation at ambient temperature, the beads were spunand washed with 1 ml of PBS three times. The washed pellet wasresuspended in SDS spl buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10%glycerol, 5% beta-mercaptoethanol and sufficient bromophenol blue toprovide visible coloration), and 30 uL of each preparation (equivalentto 15 uL of culture) was subjected to SDS-PAGE. The gel was stainedusing a Colloidal Coomassie (G-250) Staining Kit (Novex, San Diego,Calif.). A band of the predicted molecular weight of the GST-huANT3fusion protein was readily apparent, with the same intensity, in each ofthe 8 preparations from pMK3C-GST-huANT3 transformants; this band wasabsent in the control preparation.

B. Western Blot Analysis of Expression of huANT3 Fusion Proteins

E. coli transformed with either (1) pMK3A-huANT3 (the pBAD/His-huANT3expression construct) or (2) pMK3C-GST-huANT3 (the pGEX/GST-huANT3expression construct) were lysed by the addition of lysozyme (100 μg/μl;Sigma) for 20 min at room temperature, followed by one freeze/thawcycle. The negative control for the former transformant was a parallelculture of the transformed cells that had not undergone arabinoseinduction. The control for the latter transformant was a parallelculture of E. coli that had been transformed with the pGEX-4T-2 vectoronly.

Total protein concentrations of each lysate were determined using theBCA Protein Assay kit (Pierce Chemical Co.), and equivalent amounts oftotal protein from each lysate preparation were mixed with equivalentvolumes of 2× Laemmli electrophoresis buffer and subjected to SDS-PAGE.The proteins were electrophoretically transferred to nitrocellulose,which was then contacted with antibodies against the appropriate epitopeincluded in each vector (i.e., ANTI-XPRESS™ from Invitrogen forpMK3A-huANT3 and polyclonal goat anti-GST from Amersham PharmaciaBiotech, formerly Nycomed Amersham plc and Pharmacia & UpJohn Inc. forpMK3C-GST-huANT3).

In a separate experiment, the bacterial lysate from the pMK3C-GST-huANT3transformants was incubated with agarose-glutathione beads (Sigma)according to the manufacturer's instructions (see the preceding sectionand Smith et al., Expression and Purification of GlutathioneS-Transferase Fusion Proteins, Unit 16.7 of Chapter 16 in: ShortProtocols in Molecular Biology, 2nd Ed., Asubel et al., eds., John Wiley& Sons, New York, N.Y., 1992, pages 16-28 to 16-31). The beads weresuspended in Laemmli sample buffer and subjected to SDS-PAGE and Westernanalysis as described above. Although the yield of GST-huANT3 was low,perhaps because the fusion protein is inserted into the bacterialmembrane, a sufficient amount of material was recovered for theexperiment.

The results (FIG. 5) show that a specific band of the predictedmolecular weight (His-Tag+enterokinase site+antigenic site+huANT3=38kDa) was observed in the arabinose induced E.coli that were transformedwith the pBAD/his-huANT3 vector, but was absent in the non-inducedcontrol culture. Similarly, a band corresponding to GST-huANT3 wasobserved in the pMK3C-GST-huANT3-transformed E.coli, while only theunaltered GST band was observed in control E.coli transformed with theexpression vector. Purification of the GST-huANT3 fusion protein usingagarose-GSH beads produced a band of equivalent size to that observed inthe crude lysate of pMK3C-GST-huANT-transformed bacteria.

Example 3 Expression of ANT3 in Insect Cells

A. Generation of Baculovirus Expression Constructs

DNA comprising nucleotide sequences encoding huANT3 was amplified by PCRfrom a whole human brain cDNA library (Clontech) using the followingprimers. In the following representations of PCR primers, underlinednucleotides indicate sequences complementary to the 5′-ends and 3′-endsof the ANT cDNAs and double-underlined nucleotides indicate recognitionsequences for the restriction enzymes BamHI (recognition sequence:5′-GGATCC) or EcoRI (recognition sequence: 5′-GAATTC).

The PCR primers used were: Forward (sense): SEQ ID NO: 16 5′-TTATAGGATCCATGACGGAACAGGCCATCTCCTTCGCCAAA and Reverse (antisense): SEQ ID NO: 175′-TTAAAGAATTC TTAGATCACCTTCTTGAGCTCGTCGTACAG.

PCR products were digested with the restriction endonucleases BamHI (NewEngland Biolabs) and EcoRI (New England Biolabs) according to themanufacturer's recommendations. Subsequent purification was carried outby horizontal agarose gel electrophoresis and band extraction using theUltraClean GelSpin kit (Mo Bio Laboratories, Inc.).

The Baculovirus transfer vector pBlueBacHis2 (B version, Invitrogen)comprises, in 5′ to 3′ orientation, a constitutive polyhedrin promotoroperably linked to nucleotide sequences encoding (1) a translationinitiation sequence, (2) an N-terminal polyhistidine sequence, (3) anXPRESS™ epitope tag for detection and purification of the recombinantprotein and (4) an enterokinas cleavage site, followed by a multiplecloning site wherein cDNAs can be inserted.

The transfer vector pBlueBacHis2 was prepared by digestion with therestriction endonucleases BamHI and EcoRI according to themanufacturer's recommendation, and the restricted DNA was subject tohorizontal agarose gel electrophoresis and band extraction using theUltraClean GelSpin kit (Mo Bio Laboratories, Inc.). The restricted PCRproducts were ligated with the restricted expression vector DNA as inthe preceding Examples.

Competent E. coli TOP10F′ cells (Invitrogen) were transformed with theligation recation following the manufacturer's instructions. Singlecolonies were selected for growth in 3-5 ml of LB broth containing 50ug/ml ampicillin. Plasmid DNA was isolated from the bacterial culturesusing the WIZARD™Plus Series 9600 Miniprep Reagents System (Promega).

The recombinant ANT gene sequences were determined and theirauthenticities confirmed (SEQ ID NOS:1, 2 and 3 correspond to human ANTs1, 2 and 3, respectively) by DNA sequencing using the Prism Ready DyeTerminator Cycle Sequencing Kit (Perkin-Elmer, Catalog #402080) and thefollowing primers: Polyhedrin Forward Sequencing Priming Site,5′-AAATGATAACCATCTCGC (SEQ ID NO:18); Baculovirus Reverse SequencingPriming Site, 5′-ACTTCAAGGAGAATTTCC(SEQ ID NO:19); primers internal tothe ANT 3 coding sequence (sense strand), 5′-ACTTCGCCTTCACGGATA (SEQ IDNO:20); and 5′-TACGGCCAAGGGCATTCT (SEQ ID NO:21); primers internal tothe ANT 3 coding sequence (antisense strand), 5′-TGAAGCGGAAGTTCCTAT (SEQID NO:22); and 5′-ATGCCGGTTCCCGTACGA (SEQ ID NO:23). Sequence data wereanalysed using the SEQUENCE NAVIGATOR™ analysis software package(Perkin-Elmer). An isolated plasmid having the correct sequence wasnamed pMK4A-huANT3.

Although pMK4A-huANT3 contains authentic huANT3-encoding sequences, theANT3 reading frame is not synchronous with the reading frame of theHis-Tag/XPRESS™ epitope of the expression vector. Accordingly,pMK4A-huANT3 is not expected to produce recombinant ANT protein,although cells harboring it may be used as controls.

In order to generate an in-frame derivative of pMK4A-huANT3, the plasmidwas mutagenized using the QUIK-CHANGE™ Site-Directed Mutagenesis Kit(Stratagene) as in Example 1, except that the mutagenic oligonucleotideprimers used were 5′-GGCCTGTTCCGTCATCTTATCGTCATCGTCG (SEQ ID NO:24; theunderlined sequence is the reverse complement of the 5′ end of thehuANT3 reading frame), and 5′-CGACGATGACGATAAGATGACGGAACAGGCC (SEQ IDNO:25; the underlined sequence corresponds to the 5′ end of the huANT3reading frame). Several transformants were isolated, and plasmid DNApurified therefrom. The nucleotide sequences of the plasmid DNAs weredetermined and one having the “correct” sequence was identified andnamed pMK4B-huANT3.

The baculovirus expression plasmids encoding human ANT3 are referred toas “pMK4A (baculovirus shuttle, out-of-frame hu ANT3) or “pMK4A”; and“pMK4B (baculovirus shuttle, in-frame hu ANT3)” or “pMK4B”. Plasmid pM4Bhas been deposited at the American Type Culture Collection (ATCC;Manassas, Va.) on Nov. 3, 1998, and given the accession number ATCC98972.

In order to insert sequences encoding the huANT3 protein (and assoicatedregulatory sequences) into the baculovirus genome, insect cells (MAXBAC™Spodoptera frugiperda Sf9 cells, Invitrogen, Carlsbad, Calif.; orTrichoplusia ni cells, PharMingen, San Diego, Calif.) wereco-transfected with the baculoviral transfer construct pMK4B-huANT3 andlinear baculoviral (Autographa californica nuclear polyhedrosis virus,AcMNPV) DNA engineered to contain a promoterless 3′ fragment of the lacZgene (BAC-N-BLUE™, Invitrogen) using the BAC-N-BLUE™ Transfection Kit(Invitrogen) following the manufacturer's instructions. Recombinantbaculovirus plaques express functional beta-galactosidase and wereidentified as blue plaques in the presence of X-gal(5-bromo-4-chloro-3-indoyl-beta-D-glactosidase). These recombinantviruses are expression constructs that express human ANT3 polypeptide ininsect cells, as shown by the following experiments.

B. Western Blot Analysis of Baculovirus Expression Systems

High titer viral stock was produced, and recombinant protein wasexpressed in infected Sf9 (Invitrogen, Carlsbad, Calif.) or T. ni(PharMingen, San Diego, Calif.) cells according to the manufacturer'sinstructions (see also Piwnica-Worms, Expression of Proteins in InsectCells Using Baculovirus Vectors, Section II of Chapter 16 in: ShortProtocols in Molecular Biology, 2nd Ed., Asubel et al., eds., John Wiley& Sons, New York, N.Y., 1992, pages 16-32 to 16-48; Kitts, Chapter 7 in:Baculovirus Expression Protocols, Methods in Molecular Biology, Vol. 39,C. R. Richardson, Ed., Humana Press, Totawa, N.J., 1995, pages 129-142).

Transfected Sf9 cells were pelleted by centrifugation and lysed byadding 100 μl of MSB buffer (210 mM mannitol (Sigma), 70 mM sucrose(Fluka), 50 mM Tris-HCl, pH 7.4, 10 mM EDTA) and performing threefreeze-thaw cycles. A total cellular fraction, a cytosolic fraction, asubmitochondrial partical fraction, a mitochondrial fraction and aplasma membrane fraction were prepared as follows. The cell lysate wascentrifuged at 600 g for 10 minutes at 4° C. to prepare a plasmamembrane pellet. The supernatant was removed and set aside. The plasmamembrane pellet was washed with 100 ul of MSB, centrifuged at 600 g for10 minutes at 4° C., and used for the analysis. The supernatant wasremoved, combined with the first supernatant and mixed. Half of thesupernatant was used to prepare a mitochondrial fraction and a cytosolicfraction by centrifugation at 14,000 g for 15 minutes at 4° C.; thepellet represents the mitochondrial fraction and the supernatantrepresents the cytosol. The other half of the supernatant wascentrifuged at 14,000 g for 15 minutes at 4° C. to produce amitochondria-containing pellet that was resuspended in MSB, incubatedwith 0.25 mg/ml digitonin (Roche Molecular Biochemicals, formerlyBoehringer Mannheim, Indianapolis, Ind.) for 2 min and sonicated for 3min at 50% duty cycle in a cup-horn sonicator to producesubmitochondrial particles (SMPs). (See Example 13 for details regardingmitochondrial preparation from transfected T. ni cells.)

The protein content for each fraction was determined using the BCAProtein Assay kit (Pierce Chemical Co.), and 8 ug of total protein wereloaded per lane onto an SDS polyacrylamide gel, electrophoresed andtransferred to a HYBOND™ ECL Nitrocellulose Membrane (Amersham LifeScience). Fusion proteins were detected in a western blot usingANTI-XPRESS™ Antibody (Invitrogen, Catalog #R910-25) and horseradishperoxidase-conjugated anti-mouse secondary antibody (Amersham LifeScience) following the manufacturers' instructions.

The results of the Western analysis are shown in FIG. 6. RecombinantGST-huANT3 fusion protein (molecular weight 36.6 kD) was detected intotal cells, mitochondria, submitochondrial particles and the plasmamembrane. The signal was most intense in mitochondria andsubmitochondrial particles, whereas no band was detectable in thecytosolic fraction. These data suggest that the human recombinant huANT3fusion protein integrated into the mitochondrial membranes much moreefficiently than into the plasma membranes. Furthermore, all of therecombinant protein integrated into membranes since no signal wasdetected in the cytosolic fraction. The final lane of the autoradiogramshows His-tagged huANT3 isolated from cell lysates using magneticagarose beads coupled to Ni according to the manufacturers instructions(Qiagen; Hilden, Germany).

Thus, as in E. coli, huANT3 is expressed in the baculovirus/Sf9 system.Furthermore, recombinantly produced 6×His- and epitope-tagged huANT3fusion protein is appropriately localized to the mitochondria in Sf9cells despite the presence of over 35 extraneous N-terminal amino acids,and can be isolated from cellular fractions by means that take advantageof the His-Tag moiety's affinity for metals such as, e.g., nickel.

Example 4 Expression of ANT3 in Yeast

A. Expression Constructs and Host Cells

Human ANT3 cDNA was amplified by PCR as in Example 1 but using thefollowing primers. In the following representations of PCR primers,underlined nucleotides indicate sequences complementary to the 5′-endsand 3′-ends of the ANT cDNAs and double-underlined nucleotides indicaterecognition sequences for the restriction enzymes XhoI (recognitionsequence: 5′-CTCGAG) or Asp718 (recognition sequence: 5′-GGTACC).

The primers used for PCR amplification were: Forward (sense; SEQ IDNO:28): 5′-TTAATGGGTACC ATGACGGAACAGGCCATCTCCTTCGCCAAA, and Reverse(antisense; SEQ ID NO:29): 5′-TTATACTCGAGTTAGATCACCTTCTTGAGCTCGTCGTACAGG.

PCR products, and expression vector DNAs, were digested with therestriction endonucleases XhoI and Asp718 (both enzymes from RocheMolecular Biochemicals) according to the manufacturer's recommendationsusing manufacturer-supplied reaction buffers. The expression vectorpYES2 (Invitrogen) was used. This vector contains a multiple cloningsite located immediately downstream from an inducible GAL1 promoter, aswell as the 2u origin of replication and the S. cerevisiae URA3 gene forhigh-copy maintenance and selection in ura3 yeast cells, respectively.

The restricted DNAs were purified by horizontal agarose gelelectrophoresis and band extraction using the UltraClean GelSpin kit (MoBio Laboratories), ligated to each other, and used to transform E. colicells, as in the preceding Examples. Plasmid DNA was isolated fromseveral transformants, and the nucleotide sequence of the insert DNA wasdetermined and confirmed to be that of huANT3. One confirmed plastid waschosen to be used for further study and was designated pMK5A (huANT3).

A second yeast huANT3 expression vector, pMK5B, was constructed asfollows. Plasmids pMK5A and pYESTrp2 (Invitrogen) were digested withrestriction enzymes BglI and PvuII (both from New England Biolabs) andgel purified, ligated and used to transform E. coli as above. Theexpression vector pYES2Trp is similar to pYES2 but comprises a TRP1selectable marker. Plasmid DNA was isolated from several transformantsand restriction mapped to confirm the structure of the expectedexpression construct. One confirmed plasmid was chosen to be used forfurther study and was designated pMK5B (huANT3).

A third yeast huANT3 expression vector, pMK5C, was constructed using theexpression vector pYPGE2, which comprises a TRP1 selectable marker andthe strong PGK promoter upstream from a multiple cloning site (Brunelliand Pall, 1993 Yeast 9:1299-1308). Plasmid pYPGE2 DNA was digested withXhoI and Asp718, gel-purified and ligated with the XhoI- andAsp718-restricted huANT3 PCR product of Example 1. The ligation mixturewas used to transform E. coli, and plasmid DNA was isolated from severaltransformants and restriction mapped to confirm the structure of theexpected expression construct. One confirmed plasmid was chosen to beused for further study and was designated pMK5C (huANT3).

In order to generate yeast expression systems, the S. cerevisiae strainINVSc1 (MATα, his3Δ1, leu2, trp1-289, ura3-52) was transformed withpurified pMK5A, pMK5B and pMK5C DNAs using the S.c. EASYCOMP™Transformation Kit (Invitrogen). A second S. cerevisiae strain, JΔ1Δ3(MATα, ade2-1, leu2-3, leu2-112, his3-11, his3-15, trp1-1, ura3-1,can1-100, AAC1::LEU2, AAC2::HIS3, AAC3::URA3) was also transformed withthe expression constructs. The AAC genes encode the three isoforms ofthe mitochondrial ADP/ATP translocator in S. cerevisiae and areinterrupted in strain JΔ1Δ3 (Giraud et al., J. Mol. Biol. 281:409-418(1998)). It is thus expected that transformants of JΔ1Δ3, which areincapable of expressing endogenous ANT (AAC) proteins, will only expressthe human ANT protein encoded by the expression construct with whichthey have been transformed.

B. Northern Blot Analyses of Yeast Expression Systems

In order to examine levels of huANT3 mRNA production in strain JΔ1Δ3,Northern analyses of cells transformed with pMK5B and pMK5C wereperformed according to methods known in the art. In brief, transformedcells and control (untransformed) cells grown to mid-log phase,harvested and lysed. RNA was extracted from the lysates, electrophoresedand transferred to a nitrocellulose filter (see Treco, Preparation ofYeast RNA, Unit 13.12 of Chapter 13 in Short Protocols in MolecularBiology, 2nd Ed., Asubel et al., eds., John Wiley & Sons, New York, N.Y.(1992), 13:44-46 and Seldon, Analysis of RNA by Northern Hybridization,Unit 4.9 of Chapter 4, Id., 4:23-25). The XhoI- and Asp718-restrictedhuANT3 PCR product of Example 1 was radiolabelled and used as a probe,and an RNA preparation from human spleen tissue was used as a positivecontrol.

The results (FIG. 10) demonstrate the appropriately-sized ANT3-specificRNA is produced in human spleen and in yeast cells transformed itheither expression vector, but not in untransformed yeast cells. ThepYPGE2-derived expression construct pMK5C, which directs ANT3 expressionfrom the PGK promoter, clearly results in more ANT3 RNA than thepYES2Trp-derived construct pMK5B, in which ANT3 expression is driven bythe GAL1 promoter. In either case, however, significant levels ofhuANT3-specific RNA were produced in a yeast background that lacks anyendogenous adenosine nucleotide translocator proteins.

C. Western Analyses of Yeast Expression Systems

1. Production of Antibody to huANT3

As the huANT3 produced from the yeast expression constructs lacks anepitope tag, it was necessary to produce an antibody to huANT3 in orderto evaluate recombinant production of the protein. A monspecific(antipeptide) antibody specific to huANT3 was prepared as follows.

A synthetic polypeptide corresponding to a portion of huANT3 locatednear the carboxy terminus and predicted to have high antigenicityaccording to the Jameson-Wolf Index (Wolf et al., Comput. Appl. Biosci.4:187-191 (1988)) was synthesized using known means by Alpha DiagnosticInternational (San Antonio, Tex.) and determined to be at least about70% pure, preferably at least about 90% pure, by HPLC and MS analysesThe sequence of the synthetic polypeptide (SEQ ID NO:30) is:Cys-Trp-Arg-Lys-Ile-Phe-Arg-Asp-Glu-Gly-Gly-Lys-Ala-Phe-Phe

The synthetic polypeptide was conjugated to a carrier molecule, keyholelimpet hemocyanin (KLH), using MSB(m-maleimidobenzoyl-N-hydroxysuccinimide ester; Pierce Chemical Co.,Rockford, Ill.), and the conjugated material was used to immunizeseveral rabbits, according to known means (Collawn and Paterson, Units11.14 and 11.15 in Chapter 11 in: Short Protocols in Molecular Biology,2nd Ed., Asubel et al., eds., John Wiley & Sons, New York, N.Y. (1992)11:37-41. The rabbits were or are bled at 0 (preimmune, 2 mL), 7, 9, 11,13 (15 mL for each bleed) or 15 weeks (50 mL) post-inoculation. Sodiumazide (0.1%) was or is added to the bleeds as preservative.

2. Western Analyses

Western analyses of yeast expression systems are performed essentiallyas described in the preceding Examples, except that different methodsare used to prepare protein preparations from yeast cells as opposed tobacterial or insect cells. Such methods of isolating proteins from yeastare known in the art (see, for example, Dunn and Wobbe, Preparation ofProtein Extracts from Yeast, Unit 13.13 of Chapter 13 in Short Protocolsin Molecular Biology, 2nd Ed., Asubel et al., eds. John Wiley & Sons,New York, N.Y. (1992), 13:46-50). The intracellular distribution ofhuANT3 in, e.g., membrane or mitochondrial fractions, is determined asin the preceding Examples.

Example 5 Expression of ANT3 in Mammalian Cells

The preceding Examples describe a variety of means by which ANT and ANTfusion proteins can be recombinantly produced in various systems.Although such ANT proteins can be used in a variety of assays (seeinfra), it may be desirable to isolate large amounts of the native ANTprotein from mammalian cells. In particular, as described in thisExample, it may be desirable to produce recombinant viral particles inwhich ANT proteins are displayed in the viral envelope. SuchANT-displaying viral particles are expected to be very stable and usefulin a variety of assays including, for example, those in which compoundsbinding to ANT proteins are screened and identified.

Another useful outcome of mammalian expression systems is the generationand isolation of human mitochondria in which a particular ANT isoform isover-represented in order to determine the specific biological role(s)of such isoforms. For example, ANT3 is apparently ubiquitously expressedin human tissues, whereas ANT1 is primarily expressed in heart andskeletal muscle (Stepien et al., 1992, J. Biol. Chem. 267:14592-14597).Directed overexpression of huANT1 in cultured heart or muscle cells isexpected to result in mitochondria that contain mostly the ANT1 isoform.Such “ANT isoform-enriched” mitochondria can be isolated and tested forvarious mitochondrial functions.

Constructs for expressing ANT proteins in mammalian cells are preparedin a stepwise process. First, expression cassettes that comprise apromoter (and associated regulatory sequences) operably linked tonucleotide sequences encoding an ANT protein are constructed inbacterial plasmid-based systems; these expression cassette-comprisingconstructs are evaluated and optimized for their ANT-producing abilityin mammalian cells that are transiently transfected therewith. Second,the ANT expression cassettes are transferred to viral systems thatproduce recombinant proteins during lytic growth of the virus (e.g.,SV40, BPV, EBV, adenovirus; see below) or from a virus that can stablyintegrate into and transduce a mammalian cellular genome (e.g., aretroviral expression construct).

A. Transient Expression

With regards to the first step, commercially available “shuttle” (i.e.,capable of replicaton in both E. coli and mammalian cells) vectors thatcomprise promoters that function in mammalian cells and can be operablylinked to an ANT-encoding sequence include, but are not limited to, SV40late promoter expression vectors (e.g., pSVL, Pharmacia),glucocorticoid-inducible promoter expression vectors (e.g., pMSG,Pharmacia), Rous sarcoma enhancer-promoter expression vectors (e.g.,pRc/RSV, Invitrogen) and CMV early promoter expression vectors,including deriavtives thereof having selectable markers to agents suchas Neomycin, Hygromycin or ZEOCIN™ (e.g., pRc/CMV2, pCDM8, pcDNA1.1,pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo and pcDNA3.1/Hygro, Invitrogen) Ingeneral, preferred shuttle vectors for ANT genes are those havingselectable markers (for ease of isolation and maintenance of transformedcells) and inducible, and thus regulatable, promoters (as overexpressionof ANT genes may have toxic effects).

Methods for transfecting mamallian cells are known in the art (see,Kingston et al., “Transfection of DNA into Eukaryotic Cells,” Section Iof Chapter 9 in: Short Protocols in Molecular Biology, 2nd Ed., Asubelet al., eds., John Wiley & Sons, New York, N.Y., 1992, pages 9-3 to9-16). A control plasmid, such as pCH110 (Pharmacia), may becotransfected with the ANT expression construct being examined so thatlevels of ANT can be normalized to a gene product expressed from thecontrol plasmid.

Western analyses of mammalian expression systems are performedessentially as described in the preceding Examples, except thatdifferent methods are used to prepare protein preparations frommamallian cells as opposed to bacterial, insect or yeast cells. Suchmethods of isolating proteins from yeast are known in the art (see, forexample, Kingston and Sheen, Unit 9.6A and Brasier, Unit 9.6B of Chapter9 in: Short Protocols in Molecular Biology, 2nd Ed., Asubel et al.,eds., John Wiley & Sons, New York, N.Y., 1992, pages 9-17 to 9-23).Preferred expression cassettes, consisting essentially of a promoter andassociated regulatory sequences operably linked to an ANT gene ofinterest, are identified by the ability of cells transiently transformedwith a vector comprising a given ANT expression cassette to express highlevels of ANT protein when induced to do so; these expression cassettesare incorporated into viral expression vectors.

B. Viral Expression

Nucleic acids, preferably DNA, comprising preferred expression cassettesare isolated from the transient expression constructs in which they wereprepared, characterized and optimized (see preceding section). Apreferred method of isolating such expression cassettes is byamplification by PCR, although other methods (e.g., digestion withappropriate restriction enzymes) can be used. Preferred expressioncassettes are introduced into viral expression vectors, preferablyretroviral expression vectors, in the following manner.

A DNA molecule comprising a preferred expression cassette is introducedinto a retroviral transfer vector by ligation (see preceding Examples).Two types of retroviral transfer vectors are known in the art:replication-incompetent and replication-competent.Replication-incompetent vectors lack viral genes necessary to produceinfectious particles but retain cis-acting viral sequences necessary forviral transmission. Such cis-acting sequences include the Ψ packagingsequence, signals for reverse transcription and integration, and viralpromoter, enhancer, polyadenylation and other regulatory sequences.Replication-competent vectors retain all these elements as well as genesencoding virion structural proteins (typically, those encoded by genesdesignated gag, pol and env) and can thus form infectious particles in avariety of cell lines. In contrast, these functions are supplied intrans to replication-incompetent vectors in a packaging cell line, i.e,a cell line that produces mRNAs encoding gag, pol and env genes butlacking the Ψ packaging sequence. See, generally, Cepko, Unit 9.10 ofChapter 9 in: Short Protocols in Molecular Biology, 2nd Ed., Asubel etal., eds., John Wiley & Sons, New York, N.Y., 1992, pages 9-30 to 9-35.

A retroviral construct comprising an ANT expression cassette producesRNA molecules comprising the cassette sequences and the Ψ packagingsequence. These RNA molecules correspond to viral genomes that areencapsidated by viral structural proteins in an appropriate cell line(by “appropriate” it is meant that, for example, a packaging cell linemust be used for constructs based on replication-incompetent retroviralvectors). Infectious viral particles are then produced, and releasedinto the culture supernatant, by budding from the cellular membrane. Theinfectious particles, which comprise a viral RNA genome that includesthe ANT expression cassette, are prepared and concentrated according toknown methods. It may be desirable to monitor undesirable helper virus,i.e., viral particles which do not comprise an ANT expression cassette.See, generally, Cepko, Units 9.11, 9.12 and 9.13 of Chapter 9 in: ShortProtocols in Molecular Biology, 2nd Ed., Asubel et al., eds., John Wiley& Sons, New York, N.Y., 1992, pages 9-36 to 9-45.

Viral particles comprising an ANT expression cassette are used to infectin vitro (e.g., cultured cells) or in vivo (e.g., cells of a rodent, orof an avian species, which are part of a whole animal). Tissue explantsor cultured embryos may also be infected according to methods known inthe art. See, generally, Cepko, Unit 9.14 of Chapter 9 in: ShortProtocols in Molecular Biology, 2nd Ed., Asubel et al., eds., John Wiley& Sons, New York, N.Y., 1992, pages 9-45 to 9-48. Regardless of the typeof cell used, production of ANT protein is directed by the recombinantviral genome.

In a preferred embodiment, recombinantly produced ANT proteins areinserted into the cell membrane of cultured cells. Because theretroviral expression construct produces viral particles by budding ofthe cell membrane, the resultant viral particles delivered to theculture supernatant have ANT protein incorporated into their capsules,preferably on the surface of the particles. Such ANT-displaying viralparticles are expected to provide a stable format for ANT proteins andto thus be useful in assays using ANT proteins, either directly or as asource material from which ANT can be further purified. If it is desiredto minimize the amount of ANT protein inserted into mitochondrialmembranes, ρ⁰ cells, which have been treated in such a manner as to benearly or completely devoid of mitochondria, are used as host cells.

C. ANT Antisense Constructs

Antisense versions of the preceding transient and viral ANT expressionconstructs are prepared by exchanging the antisense (non-encoding)strand for a sense (ANT protein encoding) strand in a construct. SuchANT antisense constructs are useful as research reagents, i.e., toreduce levels of expression of one or more isoforms in a celltransformed or infected with such a construct in order to determine theeffects of such treatment on cellular physiology. ANT antisenseconstructs are also useful as gene therapy agents that interfere withthe translation of one or more isoforms of ANT.

Example 6 Synthesis and Properties of Representative ATR Derivatives

A number of atractyloside (ATR) derivatives were prepared for use asligands for adenine nucleotide translocators (ANTs) in the context ofhigh-throughput screening assays. These compounds bind with highaffinity (i.e., in the nM range) to ANT and are thus useful forscreening libraries of chemical compounds for molecules having highspecificity for ANT (regardless of isoform) The structure of ATR is setforth below as compound (1). Compounds (3) and (4) represent novelfluorescent derivatives of ATR, while compound (2) is an ATR derivativewhich permits introduction of the ¹²⁵I under mild conditions.

Purification

Compounds 2, 3 and 4 were purified by silica gel chromatography usingCH₂Cl₂/MeOH/AcOH (75:25:1) as the eluting solution. Detection wasachieved by staining with a 0.5% solution of vanillin in H₃PO₄/H₂O(1/1). Further purification was accomplished by reversed-phase HPLCusing a Microsorb C8 column (250×10 mm). The column was eluted at a flowrate of 2.0 mL/min with a linear gradient of methanol/acetic acid/1 Mammonium acetate 98:1:1 (“Solvent B”) and H₂O/acetic acid/1 M ammoniumacetate aqueous solution 98:1:1 (“Solvent A”). The effluent wasmonitored for absorbance at 254 nm. Compound-containing fractions werepooled, evaporated, and repeatedly co-evaporated with added methanol(3×5 mL).

Synthesis of Compound 2

Atractyloside 1 (0.10 mmol) was dried by repeated evaporation of addedpyridine (3×5 mL) and the resulting gummy residue dissolved in pyridine(5 mL). To the resulting solution, 0.20 mmol of toluenesulfonyl chloridewas added. The reaction mixture was stirred at ambient temperature for1.5 h. Then, another portion of toluenesulfonyl chloride (0.20 mmol) wasadded and the reaction left stirring an additional 1.5 h. 1 mL ofmethanol was added to the reaction mixture which was then stirred for0.5 h, after which solvents were removed by evaporation. Residualpyridine was removed by evaporation of additional methanol (5×10 mL).Silica gel chromatography followed by reversed-phase HPLC using a lineargradient of 50-80% of solvent B in solvent A for 30 min. resulted in thecompound 2 eluting at 68% solvent B. Yield: 4.3 mg, 4.9%. ESI-MS (M−H)found: 879, calc.: 879.

Synthesis of Compounds 3 and 4

7-Diethylamino-2-oxo-2H-chromene-3-carboxylic acid or 0.20 mmol of1-pyrenebutyric acid and 0.60 mmol of 1,1′-carbonyldiimidazole in 1 mLof dimethylformamide were allowed to react for 15 min. To the activatedcarboxylic acid was added a solution of atractyloside 1 in H₂O (4 mL)and the resulting reaction mixture was stirred at ambient temperaturefor 16 h. Evaporation left a gummy residue which was purified by silicagel chromatography followed by reversed-phase HPLC. Using a lineargradient of 10-80% of solvent B in solvent A for 50 min (for compound 3)or 50-100% of solvent B in solvent A for 50 min (for compound 4)resulted in compound 3 eluting at 75% B and compound 4 eluting at 82% B.Yields: compound 3, 3.1 mg, 8.0%; compound 4, 1.3 mg, 3.6%. ESI-MS (M−H)compound 3 found: 968, calc.: 968; compound 4 found: 995, calc.: 995.

Properties of Representative ATR Derivatives

As summarized in Table 1 below, compounds 3 and 4 were found to be moreadvantageous in terms of fluorescence characteristics and sensitivitycompared to the existing ATR derivatives Naphthoyl-ATR and MANT-ATR asreported by Boulay et al., Analytical Biochemistry 128:323-330,1983;Roux et al., Analytical Biochemistry 234:31-37,1996; and Lauquin et al.,FEBS Letters 67:306-311,1976. TABLE 1 Excitation Emission ExtinctionCoefficient (M⁻¹) ATR Derivative (nm) (nm) (Predicted) Naphthoyl-ATR 300405 6,200 MANT-ATR 350 460 5,800 Compound 4 341 391 17,420 Compound 3417 470 46,400

Example 7 Synthesis of Representative ATR Derivative

The further representative ATR derivative, compound 5, was prepared bythe procedure set forth below.

Synthesis of Compound 5

Dipotassium atractylate (0.10 mmol) was dissolved in 50% aq. ethanol (5mL) and palladium on charcoal (10%, 17 mg) was added to the reactionmixture. After flushing the system with hydrogen, the reaction mixturewas stirred under an atmosphere of hydrogen gas for 3 h. Removal ofcatalyst by filtration through Celite, washing with 50% aq. ethanol (10mL), and evaporation of solvents afforded a white solid. Yield afterthorough drying under high vacuum; 78.3 mg (97.3%). ESI-MS (M−2H+K)found: 765, calc.: 765. ¹H-NMR analysis confirmed the absence of alkenicprotons: DMSO-d₆) δ 0.88(d, 3H), 0.89(d, 3H), 1.02(d, 3H).

Example 8 Synthesis of Representative Iodinated ATR Derivative

Compound 2 of Example 6 may be used as intermediate for conjugation ofvariety of chemical moieties to yield further ATR derivatives. In thisexample, compound 2 is employed to introduce ¹²⁵I under mild conditionsto yield the following compound 6.

Synthesis of Compound 6

Five μl of 0.2 M sodium phosphate (pH 5) was combined with 21 ul ofNa¹²⁵I (9.25 mCi) in its shipping container (specific activity, 2100Ci/mmol; Amersham, Piscataway, N.J.). Ten ul (200 ug, 212 nmol) ofcompound 2 of Example 1 was added to the mixture. The pH was checkedwith litmus paper to confirm that it did not rise above pH 5. Themixture was allowed to stand at ambient temperature overnight (17.5hours) to yield radiolabelled compound 6. (Non-radioactive iodinated ATRderivative, for use as a “cold” competitor in binding studies, may beprepared in the same manner using unlabeled iodine). The iodinatedderivative was purified over a C18 analytical column (4×6×250 mm)(Phenomenex, Torrance, Calif.) using a 25%-55% acetonitrile gradient inrunning buffer (1% triethylammonium acetate, pH 4.5). A flow rate of 1ml/min was used to run the gradient over 30 min. The desired producteluted at 25 min. ESI-MS: 835 (M−H), 707 (m-2H-I).

Example 9 Synthesis of Representative ATR Derivatives

Activation of carboxylic acids with carbonyl diimidazole and theirreaction with ATR has been the method of choice for synthesis of various6′-O-acyl derivatives. The relatively low reactivity of the 6′-hydroxylof ATR and the presence of an allylic secondary hydroxyl in the aglyconas well as the sulfated glucose moiety, are all factors that have anegative impact on the efficiencies of these acylation reactions. Hence,yields are generally poor and the approach requires a large excess ofacylating reagents.

Two strategies for introduction of an amine functionality in the ATRsystem are described below that permit synthesis of a broader range ofATR derivatives. In the first strategy, as depicted by Scheme 1,displacement of the primary tosylate from compound 2 (Example 1) withazide followed by reduction yields the corresponding 6′-amine (compound7). Alternatively, the amine group can be introduced as part of aspacer, which permits introduction of more sterically demandingfunctional moieties. Thus, reacting the 6′-O-succinoyl derivative(compound 8; see Brandolin et al., 1974 FEBS Left. 46:149.) with amonoprotected diamine followed by deprotection affords compound 9 asillustrated by Reaction Scheme 2.

The amine-containing ATR derivatives 7 and 9 may then be reacted with avariety of fluorophors and haptens bearing reactive isothiocyanate,N-hydroxysuccinimide ester and anhydride functionalities to yield stableATR-derivatives having thiourea and amide linkages. Representative ATRderivatives that were prepared include ATR-lanthanide chelating agents(compounds 10, 11, 12, 13, 20 and 21) that have utility fortime-resolved fluorescence detection of these compounds complexed toEu³⁺. In addition, ATR was conjugated to cyanine (compounds 14 and 15)and fluorescein analogues (compounds 16 and 17) that are detectable byfluorescence with extremely high sensitivities. Coupling of biotin-NHSester with the ATR derivatives of compounds 7 and 9 provided ATR-biotinconjugates (compounds 18 and 19) that can be detected with commerciallyavailable enzyme-avidin conjugates using calorimetric, fluorescent orchemiluminescent techniques.

More specifically, a solution of compound 2 in DMF was treated withazide ion for 8 hours at 80° C. to give the 6′-azido-ATR, that waspurified by silica gel chromatography using a CH₂Cl₂/CH₃OH solventsystem supplemented with 1% acetic acid. Staudinger-reduction using 1.5equivalents of triphenylphosphine in a THF/H₂O mixture for 4 hours at RTafforded the amine of compound 7, that was isolated after silica gelchromatographic purification.

To accommodate more sterically demanding functional moieties,6′-O-succinoyl-ATR may be condensed with commercially availablemonoprotected diamines (Calbiochem-Novabiochem Corp, San Diego, Calif.)to produce ATR-mono-protected amine derivatives. Thus, EDC-mediatedcoupling of 6′-O-succinoyl-ATR in DMF with 1.1 equivalents ofmono-protected FMOC diamines yield the amide that was deprotected usingpiperidine or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in acetonitrileto furnish the ATR derivative of compound 9. The amines were purified bysilca gel chromatography as described above.

The ATR-amine derivatives of compounds 7 and 9 were coupled to a varietyof fluorophors, chelates and haptens that contained amine-reactivefunctionalities, such as isothiocyanates, anhydrides and NHS esters inaqueous DMF to generate the ATR derivatives of compounds 10 through 19:These compounds were purified by a combination of silica gelchromatography and preparative reverse phase chromatography on a C-8column using CH₃OH/H₂O gradient containing 0.1-1% acetic acid.

Example 10 Synthesis of Representative ATR Derivatives and IntermediatesTherefor

General Procedure for Coupling Atractyloside or Dihydroatractyloside toOrganic Acids

Carboxylic acid (200 μmol) and 1,1′-carbonyldiimidazole (700 μmol) weredissolved in DMF (2 mL) and stirred for 15 min. To the activated acid, asolution of atractyloside (ATR) (100 μmol, 85 mg) ordihydroatractyloside (100 μmol ) in DMF:H₂O (1:2, 6 nL) was added (in 1mL portions over ca 30 sec). The reaction mixture was stirred at roomtemperature for 60 min, after which solvents were removed by rotaryevaporation on a water bath in which the temperature was kept below 40°C. The residue was stripped of traces of DMF by repeated evaporation ofadded EtOH:H₂O (1:1, 3×20 mL). The residue was then taken up in MeOH:H₂O(1.1, 10 mL), sonicated if necessary, and filtered through a 0.45 μmfilter. Evaporation, re-dissolution in ˜1.3 mL buffer, and purificationby reverse phase HPLC furnished the desired 6′-O-acyl-ATR in yieldsranging from 5-15%.

Reverse Phase HPLC Conditions for Purification of AtractylosideDerivatives

Purification by reverse phase HPLC (RP-HPLC) was performed in a 10×250mm C-8 column, using a gradient of MeOH:AcOH:1 M NH₄OAc (buffer B,98:1:1) in H₂O:AcOH:1 M NH₄OAc (buffer A, 98:1:1). Typically, a gradientof B in A from 50-80% over 30 min was employed. For more lipophilicderivatives, the gradient was from 60-90% or 70-100% B over the sametime period.Synthesis of Dihydroatractyloside

Atractyloside, dipotassium salt (254 mg, 0.3 mmole) in 15 ml of EtOH/H₂O(1:1) was hydrogenated under atmospheric pressure for 3 hours using 51mg of activated palladium/carbon (10%) as catalyst. The catalyst wasremoved by filtration through celite and the celite bed was washed with15 ml of EtOH/H₂O. The filtrates were concentrated by rotaryevaporation, followed by drying under high vacuum overnight to providethe product as white solid (236 mg), that was pure by NMR.6′-Tosyl-Atractyloside

Atractyloside.3H₂O (255 mg. 0.3 mmole)was dried by co-evaporation withdry pyridine (3×5 ml) and then was kept under high vacuum for 16 hrs.The dried atractyloside was dissolved in 15 ml of dry pyridine and 114mg of tosyl chloride (0.6 mmole) was added. The reaction was stirred for2 hrs at 23° C. and then an additional 114 mg of tosyl chloride wasadded and the reaction was allowed to continue for an additional 1.5hrs. Methanol (1 ml) was added to the reaction mixture to scavengeexcess tosyl chloride and the mixture was stirred for several minutes.The mixture was evaporated to dryness, and residual pyridine was removedby evaporation of added methanol (2×30 ml). The crude product waspartially purified by silica gel flash chromatography (CH₂Cl₂/MeOH, 3:1with 1% AcOH). The product containing fractions were dissolved in 3 mlof H₂O:MeOH:1M NH₄OAc:HOAc (49:49:1:1) and purified by RP-HPLC using a50%-80% gradient of MeOH:AcOH:1 M NH₄OAc (buffer B, 98:1:1) inH₂O:AcOH:1 M NH₄OAc (buffer A, 98:1:1). Product containing fractionswere pooled, evaporated, and subjected to additional co-evaporation of3×10 ml of MeOH. Tosyl-atractyloside was obtained as a glassy materialweighing 60 mg.General Procedure for Lactoperoxidase-Catalyzed Iodination of4-Hydroxyphenyl Derivatives

To a solution of 4′-hydroxy-biphenyl-4-carboxylic acidatractylosid-6′-yl ester (1.0 mg, 1.0 mmol) in H₂O (120 μL) were addedaqueous solutions of lactoperoxidase (50 μL, 200 IU/mL), NaI (10 μL, 100mM) and H₂O₂ (20 μL, 100 mM). The reaction was left at room temperaturefor 1 h after which it was frozen at −20° C. The next day (˜20 hourslater) the reaction mixture was thawed and subjected to RP-HPLC. Threemajor peaks were eluted and electrospray ionization-mass spectrometic(ESI-MS) analysis confirmed their identity as unreacted startingmaterial, and monoiodinated, and diiodinated atractyloside derivatives.

These conditions can be modified to drive the reaction completely to themono and di-iodinated forms with additional aliquots of NaI and/orenzyme.General for Synthesis of Iodophenols

The iodination procedure described in Acta Chem, Scand. 12, 188 (1958)was used for mono-iodination of 3-(4-hydroxyphenyl)propionic acid,4′-hydroxy-4-biphenylcarboxylic acid and 4-hydroxybenzoic acid. Thismethod is also applicable for the mono-iodination of3-(4-methoxyphenyl)-propionic acid and di-iodination of3-(3-iodo-4-hydroxyphenyl)propionic acid.

Thus a solution of KI (1.99 gm, 12 mmole) and iodine (1.22 gm, 4.8mmole) in 20 ml of H₂O was added in a dropwise fashion to a solution of3-(4-hydroxyphenyl)propionic acid (0.83 gm, 5 mmole) in 100 ml ofconcentrated aqueous ammonia solution over 20 min. The reaction mixturewas stirred for an additional 40 min and then subjected to vacuum toremove the ammonia. The mixture was dried further by rotary evaporationto afford an oily residue. The crude material was partitioned between 2MHCl (50 ml) and ether (2×50 ml) and the ether layers were combined andconcentrated to give a yellowish solid residue. Flash silica gelchromatography using 95:5 CH₂Cl₂/MeOH as eluant, concentration ofproduct containing fractions and recrystallization in 1:1 benzene hexaneafforded 790 mg of 3-iodo-4-hydroxyphenylpropionic acid.

3-(3,5-Diiodo-4-hydroxyphenyl)propionic acid was prepared in similarfashion using 5.2 equivalents of KI and I₂. equivalents of 12. Followingcrystallization from toluene, the di-iodo derivative was obtained in 77%yield.5-iodo-6-hydroxy-2-naphthoic acid

Mono-iodination of 6-hydroxy-2-naphthoic acid and 4-hydroxybenzoic acidwas carried out following the procedure of Edgar and Falling (J. Org.Chem. 55, 5287, 1990). Thus, 0.75 gm of (4.34 mmol) was dissolved in 19ml of MeOH and 1.04 gm of NaI (and 0.27 gm of NaOH was added. Thesolution was cooled to 0□ C. and aqueous NaOCl (4% solution, 12.9 ml)was added dropwise over 75 min. The resulting mixture was stirred for 1hr at 0° C. and then treated with 7 ml of 10% aqueous sodiummetabisulfite. The mixture was adjusted to pH 7 using 5% HCl andextracted with 40 ml of ether. The organic layer was washed with brineand dried over MgSO₄. The solution was concentrated to an off-whitesolid, that was recrystallized from toluene/CH₃OH to provide 0.42 gm of5-iodo-6-hydroxy-2-naphthoic acid.Reaction of 6′-Tostylatractyloside with 1,2-ethylenediamine

6′-Tosyl-Atractyloside (25 mg) was dissolved in 2 ml of1,2-ethylenediamine and the mixture was stirred at 23° C. overnight. The1,2-ethylenediamine was removed in vacuo, the residue was dissolved inMeOH/H₂O (2:1) and 10.6 mg of the product was isolated in by RP-HPLCusing the conditions described above. Proton nmr and mass spectraindicate the loss of the isovaleryl group.

Reaction of N-(6-Deoxy-apo-atractylosyl)-ethanediamine with BoltonHunter Reagent

N-(6-Deoxy-apo-atractylosyl)-ethanediamine (10.6 mg) in 2.75 ml ofDMF/DMSO (8:3) was reacted with 60 mg of4-hydroxyphenylpropionyl-N-hydroxysuccinimidyl ester (Bolton Hunterreagent) at 23° C. for 16 hrs. The solution was diluted with water andpurified by RP-HPLC to afford 9.1 mg of the desired compound.

Lactoperoxidase catalyzed iodination of the compound leads to aquantitative conversion to the di-iodo-apo-atractyloside derivative.Reaction of Atractyloside with Succinic Anhydride

Atractyloside.3H₂O (255 mg. 0.3 mmole) was dried by co-evaporation withdry pyridine (3×5 ml) and then was kept under high vacuum for 16 hrs.The dried atractyloside was dissolved in 6 ml of dry pyridine and 60 mgof succinic anhydride (0.6 mmole) was added. The mixture was kept at 80°C. for 30 min, another 60 mg (0.6 mmole) of succinic anhydride was addedand the reaction mixture was stirred for an additional 3 hrs. Thepyridine was removed in vacuo, and the residue was triturated with 10 mlof MeOH. The 6′-O-succinyl-ATR derivative was collected by filtration asa white solid, washed with MeOH and dried overnight over P₂O₅.Reaction of 6′-O-succinylatractyloside with Tyramine and Iodination

6′-O-Succinylatractyloside (85 mg) was dissolved in 2 ml of DMF and 28mg of tyrarnine and 100 mg of PyBOP was added. The mixture was stirredat 23° C. for 16 hrs. The crude mixture was subjected to RP-HPLC and thedesired amide was isolated in 19.7 mg. Lactoperoxidase catalyzediodination of the product using the standard conditions described aboveprovides the mono and the di-iodinated products.Synthesis of 4-(4-Hydroxy-3-Methyl)-Butyric Acid

To 4 gm (30 mmol) of AlCl3 in 50 ml of 1,2-dichloroethane at 0° C. wasadded 2.7 gm (27 mmol) of succinic anhydride and the mixture was stirredfor 20 min. 2-Methylanisole (3.1 ml, 25 mmol) was added, and thereaction mixture was warmed to 23° C. and stirred for 12 hrs. Themixture was poured into 300 ml of ice-cold water and the precipitate wasfiltered off. The precipitate was washed with 2×300 ml of water toafford a white solid. The solid material was dried under vacuum toafford 3.51 gm of product that was used in the next reaction.

3-(4-Methoxy-3-methylbenzoyl)propionic acid (4.4 gm, 20 mmol) and 4.49gm of KOH pellets (80 mmol) were dissolved in 30 ml of ethylene glycoland 3.88 ml of hydrazine hydrate (80 mmol) was added to the stirredsolution in four portions. The resulting reaction mixture was heated at155° C. for 24 hrs in an oil bath. After cooling, the reaction mixturewas taken up in 100 ml of benzene and washed with 10% aqueous citricacid. The organic layer was washed with another portion of citric acid,dried over anhydrous sodium sulfate, and concentrated in vacuo to affordan oily residue that crystallized upon standing. The material wastriturated with hot hexane and the solvent was evaporated off to afford3.55 gm of a crystalline solid that was homogeneous by silica gel tlcusing hexanelethyl acetate (8:2) as eluting solvent.

4-(4-Methoxy-3-methylphenyl)butyric acid (3.12 gm) was heated in 120 mlof a 1:1 mixture of 48% aqueous HBr/acetic acid at 155° C. for 24 hrs.The reaction mixture was cooled to room temperature and was extractedwith 200 ml of benzene/ether (1:1). The organic layer was dried overanhydrous magnesium sulfate and concentrated to afford a light brownsolid residue. The reaction products were separated by silica gel flashchromatography using hexane/ethyl acetate (3:1) as eluting solvent toprovide 0.86 mg of 4-(4-hydroxy-3-methyl)-butyric acid as a light yellowsolid.

Representative Synthesis of Atractyloside Derivatives

To a solution of 3-(4-hydroxyphenyl)propionic acid (HPP) (0.498 g, 3.0mmole) in 10 ml of anhydrous DMF was added carbonyldiimidazole (0.486 g,3.0 mmole). The mixture was stirred at room temperature for 30 minutesand added in portions (2 ml/hour) to a solution of atractyloside (ATR)(0.086 g, 0.1 mmole) in 1 ml of anhydrous DMF. The reaction mixture wasstirred at room temperature overnight and quenched with 1 ml of water.The solvent was evaporated under vacuum and the residue was dissolved inethyl acetate (75 ml) and water (50 ml). The aqueous layer is separated,extracted with ethyl acetate (3×75 ml) and concentrated under vacuum.The residue was dissolved in 1.5 ml of metbanol/water(1/1), filteredthrough a 0.2 mm filter and purified using HPLC with a preparative C-8column (microsorb, 10×250 mm) using a linear gradient elution of 30%-60%solvent B with a flow rate of 2.0 ml/min (solvent A: H2O/HOAc/NH4Oac(1.0M aq.): {1000/1/1}; solvent B: CH3OH/HOAc/NH4OAc(1.0 M, aq.):{1000/1/1}). The title compound (compound 36 of Example 11 below) wasobtained as a white film (6.2 mg).

Example 11 Further Representative ATR Derivatives

Following the procedures set forth in Example 10, the following ATRderivatives were prepared.

Atractyloside Derivatives

Cpd R MS NMR 22

 879.3 (M—H)⁻ 799.3 (M—SO₃—H)⁻ ¹H NMR (CD₃OD) δ 0.63(s, 3H), 0.95(d,3H), 0.96(d, 3H), 4.94(s, 1H), 5.09(s, 1H), 7.53(m, 2H), 7.60(m, 1H),7.93(d, 1H), 8.07(d, 1H), 8.26(d, 1H), 8.88(d, 1H) 23

1083 (M—H)⁻1003 (M—SO₃—H)⁻ ¹H NMR (CD₃OD) δ 0.65(s, 3H), 0.95(d, 3H),0.96(d, 3H), 5.02(s, 1H), 5.13(s, 1H), 6.61 (ddd, 2H), 6.69(d, 2H),6.86(dd, 2H), 7.34(d, 1H), 8.33(dd, 1H), 8.67(s, 1H) 24

 999 (M—H)⁻ ¹H NMR (CD₃OD) δ 0.94(s, 3H), 0.95(d, 3H), 0.96(d, 3H),5.05(s, 1H), 5.15(s, 1H), 6.75(d, 1H), 7.04(dd, 1H), 7.52(d, 1H) 25

 921 (M—H)⁻ 841 (M—SO₃—H)⁻ ¹H NMR (CD₃OD) δ 0.75(s, 3H), 0.95(d, 3H),0.96(d, 3H), 4.97(s, 1H), 5.08(s, 1H), 6.89(d, 2H), 7.52(d, 2H), 7.64(d,2H), 8.07(d. 2H) 26

1047 (M—H)⁻ 967 (M—SO₃—H)⁻ ¹H NMR (CD₃OD) δ 0.73(s, 3H), 0.95(d, 3H),0.96(d, 3H), 4.98(s, 1H), 5.09(s, 1H), 6.93(d, 1H), 7.52(dd, 1H),7.61(d, 2H), 7.98(d. 1H), 8.08(d, 2H) 27

— ¹H NMR (CD₃OD) δ 0.81(s, 3H), 0.95(d, 3H), 0.96(d, 3H), 5.04(s, 1H),5.15(s, 1H), 6.80(d, 2H), 7.90(d, 2H), 7.64(d, 2H) 28

1097.1 (M—H)⁻1017.0 (M—SO₃—H)⁻ ¹H NMR (CD₃OD) δ 0.83(s, 3H), 0.95(d,3H), 0.96(d, 3H), 5.03(s, 1H), 5.15(s, 1H), 6.84(d, 1H), 7.88(dd, 1H),8.33(d, 1H) 29

1043.2 (M—2H + Na)⁻1021.2 (M—H)⁻ ¹H NMR (CD₃OD) δ 0.66(s, 3H), 0.95(d,3H), 0.96(d, 3H), 4.93(s, 1H), 5.05(s, 1H), 7.22(d, 1H), 7.87(d, 1H),8.04(dd, 1H), 8.08(d, 1H), 8.50(d, 1H) 30

 944(M—H)⁻ 864(M—SO₃ —H)⁻ ¹H NMR (CD₃OD) δ 0.97(s, 3H), 0.98(d, 6H),5.06(s, 1H), 5.17(s, 1H), 5.70(d, 2H), 7.03(d, 2H). 31

1070(M—H)⁻ 990(M—SO₃-H)⁻ — 32

1116(M—SO₃—H)⁻ — 33

1125(M—H)⁻1147(M—2H + Na)⁻ ¹H NMR (CD₃OD) δ 0.95(s, 3H), 0.96(s, 6H),5.05(s, 1H), 5.15(s, 1H), 7.58(s, 2H) 34

 923.2 (M—2H + Na)⁻ 901.3 (M—H)⁻ — 35

1049.3 (M—2H + Na)⁻ — 36

 873(M—H)⁻ 895(M—2H + Na)⁻ ¹H NMR (CD₃OD) δ 0.94(s, 3H), 0.96(d, 6H),5.05(s, 1H), 5.16(s, 1H),

Dihydroactractyloside Derivatives

Cpd R MS NMR 37

 897 (M—2H + Na)⁻ 875 (M—H)⁻ ¹H NMR (CD₃OD) δ 0.93(s, 3H), 0.95(d, 3H),0.96(d, 3H), 1.08(d, 3H), 6.69(d, 2H), 7.02(d, 2H), resonances fromalkenic protons absent 38

1105 (M—2H + Na)⁻1083 (M—H)⁻ ¹H NMR (CD₃OD) δ 0.93(s, 3H), 0.95(d, 3H),0.96(d, 3H), 1.08(d, 3H), 6.74(d, 1H), 7.05(dd, 1H), 7.53(d, 1H),resonances from alkenic protons absent

Apoactractyloside Derivatives

Cpd R MS NMR 39

 831(M—H)⁻ ¹H NMR (CD₃OD) δ 1.00(s, 3H), 5.07(s, 1H), 5.17(s, 1H),6.69(d, 2H), 7.03(d, 2H) 40

1083(M—H)⁻1105(M—2H + Na)⁻

Example 12 Binding Assays Using Recombinant huANT3

A. GST-huANT3 Recombinantly Produced in E. coli

Following arabinose induction, transformed or sham transformed (vectoronly) E. coli were collected by centriflugation at 2000 g for 10 min.The bacterial pellets were resuspended in MSB, to which lysozyme (100μg/ml) was added. After 20 min at room temperature, the lysates weresubjected to one freeze-thaw cycle followed by sonication as describedabove. The resultant membrane preparation was used for binding assays.

To estimate maximal binding and the extent of overexpression of thehuANT3, 25 μg of membrane protein was incubated with varying amounts of[³²P]ATP (1-500 μM) in binding buffer (120 mM KCl, 10 mM Tris, 1 mMEDTA, pH 7.4) for 2 hr at room temperature. The membranes with bound ATPwere sedimented by centrifugation at 5000 g for 5 min, and washed oncewith binding buffer. Membrane pellets were then mixed with 5 mlscintillation cocktail and counted. The results are presented in Table2. TABLE 2 Saturation Binding of [³²P]ATP to E. coli Membranes cpm boundby: [ATP], uM Sham Transfomed Cells huANT3-Producing Cells 0.1 109 1910.5 95 49 1.0 147 325 5.0 123 N.D. 10 214 263 50 549 2,727 100 718 5,772500 2,140 9,715N.D., not determined

The data presented in Table 2 indicate that the affinity of the ATPbinding was ˜6 μM. ATP binding was completed abolished by the additionof atractyloside (10 μM) to the assay. These results support thecontention that the measured ATP binding was predominantly torecombinantly produced ANT3 protein.

Agarose-glutathione beads were incubated with solubilized (using Dnase,Rnase and 0.1% Triton X-100; see Example 1, section D) bacterial lysate(see Example 2), and substituted for the E. coli membranes in bindingassays. Best results (i.e., more specific binding) were seen when thebeads were preincubated with bovine serum albumin (BSA, 0.1%) SpecificATP binding (1,070 cpm) was also observed in this experiment (compare tononspecific binding of 279 cpm in the presence of 10 mM non-radiolabeledATP).

B. huANT3 from a Sf9/Baculovirus Expression System

Sf9 cells expressing huANT3 were grown in spinner flasks. The cells wereharvested by centrifugation at 2,000 g for 5 min. The cell pellet wasresuspended in MSB and subjected to 3 freeze-thaw cycles. Cell membranesand debris were removed by centrifugation at 600 g for 5 min;mitochondria were collected by centrifuging the supernatant at 20,000 gfor 15 min. The mitochondrial pellets were suspended in MSB, and usedfor binding assays as described above. Homologous competition of[³²P]ATP binding was performed using 25 μg mitochondrial protein perassay.

As illustrated in FIG. 7, ATP bound to the mitochondria with Kd=13 μM avalue consistent with ATP binding to ANT. Furthermore, the ATP bindingwas displaced by low concentrations of atractyloside (FIG. 8).Homologous competition binding assays using [¹²⁵I]atractyloside revealedspecific binding with Kd=12 nM (FIG. 9). These findings are consistentwith the presence of functional huANT3 in the mitochondrialpreparations.

His-tagged huANT3 protein was purified from baculovirus-infected Sf9solubilized cell lysates using Ni-agarose magnetic beads; Sf9 cells thathad not been infected were used as negative controls. The beads wereincubated with [³²P]ATP (1 or 100 μM) for 2 hr. The beads were washedand then counted to determine the amount of bound ATP. As shown in Table3, the [³²P]ATP binding was significantly higher in material recoveredfrom the infected cells than in the controls. Binding saturation hadessentially been achieved with 1 μM ATP. TABLE 3 Binding of [³²P]ATP toPurified His-Tagged huANT3 cpm bound by: [ATP], uM Control (Uninfected)Cells huANT3-Producing Cells 1.0 43 149 100 30 160

Example 13 Competitive Binding Assays

Atractyloside analogs (Example 6; Table 1; see also Examples 7-9) wereused in pseudo-homologous competition binding assays using Sf9/huANT3mitochondria. Mitochondria (see Example 12, 25 μg/tube) were incubatedwith 0.5 nM [¹²⁵I]atractyloside and varying concentrations ofnon-radiolabeled atractyloside or fluorescent atractyloside derivativesas described above (FIGS. 7-9).

The results (Table 4) show that three of the atractyloside derivatives(MANT-, Pyrene- and Coumarin-atractyloside) had relative bindingaffinities similar to that of authentic atractyloside (IC₅₀<500 mMrelative to atractyloside). Each of these derivatives is fluorescent,and may therefore be useful as detectable ligands for binding assays.TABLE 4 Competitive Binding Assays Using [¹²⁵I]Atractyloside cpm[¹²⁵I]Atractyloside bound in the presence of: [ATR Derivative], nM ATRCOU-ATR PYR-ATR MANT-ATR 0.0 227 437 437 437 1.0 224 391 350 — 5.0 146 —— — 10 42 371 349 229 50 26 — — — 100 — 277 362 195 1,000 36 174 238 —5,000 45 — — — 10,000 0 100 0 —Abbreviations and symbols:ATR, atractlyoside.COU-ATR, Coumarin-atractyloside, (Table 1, compound 3).PYR-ATR, Pyrene-atractyloside, (Table 1, compound 4).MANT-ATR, MANT-atractyloside, (Table 1, Roux et al. 1996 Anal. Bioch.234:31)—, not determined.

Atractyloside analogs (Example 6; Table 1; see also Examples 7-9) werealso used in pseudo-homologous competition binding assays using T.ni/huANT3 mitochondria or bovine mitochondria. Mitochondria fromnoninfected T. ni cells, or T. ni cells infected with a baculovirusexpressing huANT3 (see Example 3) were prepared as follows: T. ni cellswere prepared by a subcontractor (PharMingen, San Diego, Calif.) asportions of about 250 mg of cells per tube. Each portion was suspendedin 1 ml of MSB with protease inhibitors (leupeptin, final concentration10 ug/ml; pepstatin, final concentration 10 ug/ml; aprotinin, finalconcentration, 2 ug/ml; phenylmethylsulfonyl fluoride, [PMSF], finalconcentration, 100 μM; all from Sigma Chemical Co., St. Louis, Mo.). Theresuspended cell suspensions were frozen and thawed twice, thenhomogenized using a rotating teflon-coated probe and a close-fittingglass container (10 passes). The cellular homogenate was centrifuged(3,700 rpm, approximately 1,500×g) at 4° C. for 5 minutes; thissupernatant from the first spin was saved. The pellet was washed withabout 500 μl of MSB with protease inhibitors, centrifuged (3,800 rpm,approximately 1,600×g) at 4° C. for 5 minutes, and supernatant from thisspin was combined with the supernatant from the first spin. The combinedsupernatant was centrifuged (14,000 rpm, approximately 20,800×g) at 4°C. for 15 minutes, and the pellet was resuspended in 300 μl of a 1:1solution of (a) 20 mM MOPS and (b) MSB, wherein both (a) and (b) containthe previously described protease inhibitors. The resultant suspensionwas frozen and thawed three times.

Bovine mitochondria were prepared as follows: Essentially all of the fatand cholesterol in clogged arteries was removed from two bovine heartswhich were then cut into 1-inch cubes. The cubes were ground in a meatgrinder using the fine setting. Three hundred (300) gm portions of theground heart were weighed out and, to each was added 400 ml of IsolationBuffer (IB; 250 mM sucrose, 1 mM sodium succinate, 0.2 mM K⁺ EDTA, 10 mMTris-base, pH 7.8). (All buffers were filter sterilized, and columnbuffers were degassed, and, unless otherwise noted, all steps werecarried out at 0 to 4° C. on ice or in pre-cooled rotors andcentrifuges.) The preparations were mixed in a blender two times for 15seconds on high setting and, in between and after blends, the pH wasadjusted to 7.8 with 2M Tris-base. The homogenate was centrifuged for 20minutes at 1,200×g, and the supernatant was poured through two layers ofcheese cloth and adjusted to pH 7.8 with 2M Tris-base. The supernatantwas then centrifuged for 30 minutes at 11,000×g. The supernatant wasdecanted, and the buff-colored outer pellet was dislodged with about 10ml of IB and discarded. The brown inner pellet (heavy mitochondria) wasresuspended in IB (about 10 ml per pellet). The pellets were homogenizedin a glass-teflon homogenizer (2 passes at high drill speed). Sampleswere combined and centrifuged for 30 minutes at 11,000×g. Thesupernatant was decanted, and the pellets were resuspended in 60 ml ofIB per 900 gm of ground heart This centrifugation step was repeated andthe pellets were finally resuspended in IB (60 ml per 900 gm of groundheart). One kilogram of beef heart typically yields about one (1) gramof mitochondria.

The mitochondrial preparations were divided into aliquots (typically, 50μl for T. ni mitochondria or 20 μl for bovine mitochondria) and theneither used directly in assays or flash frozen and stored at −80° C. Thetotal protein content in the mitochondrial preparations was determinedusing the enhanced protocol (30 minutes at 60° C.; seehttp://www.ruf.rice.edu/˜bioslabs/methods/protein/BCA.html) of thebicinchoninic acid (BCA) assay (available in kit form from Pierce,Rockford, Ill.).

In the “Tube Assay,” mitochondria (from about 1 to 10 μg of totalprotein) were resuspended in 100 μl of Tris-KCl buffer with 0.1% BSA, pH7.4. ¹²⁵I-labeled compound 24 (Example 11) was added to a finalconcentration of 0.5 nM. When used, competitors were added at theseconcentration ranges: unlabeled atractyloside or compound 24, finalconcentration from 5 nM to 10 μM; unlabeled ADP (a lower affinitycompetitor) was added at a final concentration of 500 nM to 1 mM.

The reaction mixes were incubated on ice for 60 minutes and thenpelleted by centrifugation (approximately 16,000×g) for 11 minutes at 4°C. Unbound ¹²⁵I-compound 24 was removed by aspiration. The pellets werecontact-washed with Tris-KCl buffer, pH 7.4, and recentrifuged. Theresultant pellets were aspirated and the radioactivity (dpm) in each wasdetermined by gamma counting.

Representative results are shown in FIGS. 11-18. The data presented inFIGS. 11 and 12 show that mitochondria (5 μg of protein/tube) from bothbeef heart (FIG. 11) and T. ni cells expressing huANT3 (FIG. 12)specifically bind ¹²⁵I-compound 24 in a manner that is inhibited byincreasing concentrations of unlabeled compound 24, but, as expected,little or no binding is seen when mitochondria are excluded from thereaction mixes.

FIGS. 13 and 14 show competitive inhibition of ¹²⁵I-compound 24 bindingto mitochondria (1 μg of protein/tube) from beef heart (FIG. 13) and T.ni cells expressing huANT3 (FIG. 14) by compound 24 that is notdetectably labeled, unlabeled atractyloside (ATR), and unlabeledadenosine diphosphate (ADP). In both instances, ATR and compound 24yield comparable competition curves, although ATR appears to have aslightly higher affinity than compound 24. However, both ATR andcompound 24 bind with much higher (about 1,000 fold) affinity than thelow affinity ANT ligand ADP.

FIG. 15 shows the competitive inhibition, by unlabeled ATR, of bindingof ¹²⁵I-compound 24 to mitochondria (1 μg of protein/tube) from T. nicells expressing huANT3 and control T. ni cells (i.e., non-infected T.ni cells). As shown in FIG. 15, there was only slight inhibition of¹²⁵I-compound 24 binding to control (nontransformed) mitochondria byhigher concentrations of ATR. In contrast, binding of ¹²⁵I-compound 24to mitochondria from T. ni cells expressing huANT3 was increasinglyinhibited by higher concentrations of ATR.

FIG. 16 shows competitive inhibition, by unlabeled compound 24 and bybongkrekic acid (BKA), of ¹²⁵I-compound 24 binding to mitochondria (1 μgof protein/tube) from beef heart. BKA effectively displaced labeledcompound 24, albeit with a slightly lower affinity than unlabeledcompound 24. FIG. 17 shows competitive inhibition of ¹²⁵I-compound 24binding to beef heart mitochondria by either of the ATR derivatives,compound 23 (see Example 11) and compound 28 (see Example 11). As shownin FIG. 17, ATR exhibited an IC₅₀ of approximately 44 nM compound 23 anIC₅₀ of approximately 105 nM, and compound 28 an IC₅₀ of approximately695 nM.

In FIG. 18, data are presented depicting competitive inhibition of¹²⁵I-compound 24 binding to beef heart mitochondria by the ATRderivative compound 5 (see Example 7). As shown in FIG. 18, the IC₅₀ forcompound 5 was approximately 3.3 μM.

Competitive binding assays were also performed using recombinantHis-tagged huANT3 (see Example 3) immobilized on Ni beads (FIG. 19)instead of mitochondria. To prepare the bead-immobilzed huANT3,mitochondria from T. ni cells infected with a baculovirus expressinghuANT3 (see Example 3) were solubilized with 0.5% O-glucopyranoside inthe presence of 0.5 nM ¹²⁵I-compound 24, Ni-agarose beads (Qiagen,Hilden, Germany), and various concentrations of ATR or BKA as unlabeledcompetitors. After 1 hour at 4° C., the beads were washed andradioactivity that remained associated with the beads was counted.(Background binding of [¹²⁵I] compound 24 to Ni-agarose beads (Qiagen)in the absence of His-tagged huANT3 was approximately 700-800 cpm andwas not subtracted from the radioactivity shown in FIG. 19.) The results(FIG. 19) show that both ATR and BKA effectively compete with compound24 in a manner similar to that observed in assays using intactmitochondria (cf. FIGS. 14 and 15).

Example 14 High Throughput Screening Assay for Compounds Targeted to ANTProteins and Polypeptides

The recombinantly produced ANT proteins, ANT fusion proteins anddetectably labeled ANT ligands described herein are incorporated intoautomated assay systems. Such automated systems are useful for highthroughput screening (HTS) of candidate ANT-binding compounds orchemical libraries comprising such compounds. Such compounds may befurther characterized and developed as drug candidates and drugs usefulfor preventing, treating or curing diseases or disorders resulting fromthe overexpression or dysfunction of one or more ANT proteins or fromthe overexpression or dysfunction of a factor that positively regulatesor stimulates ANT proteins.

A preferred element of many automated assay systems is the incorporationof a target molecule (in the present instance, an ANT protein) into a96-well plate. This format is readily adaptable for use in a variety ofautomated label detection systems. For HTS assays, robotic labeldetection systems are preferred.

As one example of an HTS comprising the elements describes herein, theGST-huANT3 fusion protein of Example 2 is contacted with REACTI-BIND™glutathione-coated 96-well plates (Pierce). Glutathione coatedstrip-well plates are preferably used for assays comprising radiolabeledANT ligands (e.g., iodinated atractyloside derivates; see Example 7),whereas black opaque glutathione coated 96-well plates are preferred forassays comprising fluorescent ANT ligands (such as are described in,e.g., Examples 6-9); both types of glutathione coated plates arecommercially available (Pierce).

In a typical assay, 1 to 50 ug of GST-huANT3 protein (i.e., totalsolubilized protein prepared as in Example 2) is added pergluthathione-coated well to each well of a 96-well plate. lodinatedatractyloside derivate (¹²⁵I-ATR) is added to the wells (0.5 nmol/well).In a control experiment, unlabeled atractyloside (ATR; Sigma) is used asa ‘mock’ drug at a concentration of from about 1 to about 10,000 nM.That is, unlabeled ATR is used to displace a labeled atractylosidederivative (e.g., ¹²⁵I-ATR). Unlabeled ATR thus acts as a positivecontrol for an HTS in which various compounds are screened for theirability to displace a labeled ANT ligand.

As an example of the automated label detection systems used in the HTSassays of the Example, when the detectably labeled ANT ligand of theassay is ¹²⁵I-ATR, an automatic gamma counter is used. Alternatively,¹²⁵I-ATR can be used in scintillation proximity assays (SPA). Forexample, a GST-huANT fusion protein is contacted with ScintiStrip96-well plates coated with glutathione (EG&G Wallac). The polystyrene ofthese plates contains a scintillating agent that emits beta radiationwhen excited by a gamma emitter in close proximity thereto. The betaradiation is then detected by any appropriate automatic beta counter.When fluorescent ANT ligands are used in the HTS assay, an automaticfluorescence counter is used and may be, for example, a FLUOROCOUNT™Counter (Packard Instrument Company, Meriden, Conn.).

1-57. (Canceled).
 58. A method for determining the presence of a humanadenine nucleotide translocator (ANT) polypeptide in a biological samplecomprising: contacting a biological sample suspected of containing ahuman ANT polypeptide with an ANT ligand under conditions and for a timesufficient to allow binding of the ANT ligand to a human ANTpolypeptide; and detecting the binding of the ANT ligand to a human ANTpolypeptide, and therefrom determining the presence of the human ANTpolypeptide in said biological sample, wherein the ANT ligand comprisesan atractyloside derivative having the following structure:

and stereoisomers and pharmaceutically acceptable salts thereof, whereinR₁ is halogen, —OC(═O)R₄ or —NHR₄; R₂ is hydrogen or —C(═O)R₅; R₃ is—CH₃ or ═CH₂; R₄ is -X-arylalkyl, -X-substituted arylalkyl,X-heteroaryl, or -X-heteroarylalkyl, wherein X is an optional amido oralkylamido linker moiety; and R₅ is alkyl. 59-63. (Canceled).
 64. Themethod of claim 58 wherein the atractyloside derivative is detectablysubstituted at the 6′ hydroxyl to form a detectable atractylosidederivative.
 65. The method of claim 64 wherein the detectableatractyloside derivative comprises a radioloabeled substituent.
 66. Themethod of claim 65 wherein the radiolabeled substituent is selected fromthe group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C and ³⁵S.
 67. The method ofclaim 64 wherein the detectable atractyloside derivative comprises afluorescent substituent.
 68. The method of claim 67 wherein the ANTligand further comprises a Eu³⁺ atom complexed to the atractylosidederivative.
 69. The method of claim 64 wherein the detectableatractyloside derivative comprises covalently bound biotin.
 70. Themethod of claim 58 wherein wherein R₁ is —NHR₄.
 71. (Canceled).
 72. Amethod for isolating a human adenine nucleotide translocator (ANT)polypeptide from a biological sample, comprising: contacting abiological sample suspected of containing a human ANT polypeptide withan ANT ligand under conditions and for a time sufficient to allowbinding of the ANT ligand to the human ANT polypeptide; and recoveringthe human ANT polypeptide, and thereby isolating human ANT from abiological sample, wherein the ANT ligand comprises an atractylosidederivative having the following structure:

and stereoisomers and pharmaceutically acceptable salts thereof, whereinR₁ is halogen, —OC(═O)R₄ or —NHR₄; R₂ is hydrogen or —C(═O)R₅; R₃ is—CH₃ or ═CH₂; R₄ is -X-arylalkyl, -X-substituted arylalkyl,X-heteroaryl, or -X-heteroarylalkyl, wherein X is an optional amido oralkylamido linker moiety; and R₅ is alkyl.
 73. The method of claim 72wherein the ANT ligand is covalently bound to a solid phase.
 74. Themethod of claim 72 wherein the ANT ligand is non-covalently bound to asolid phase. 75-112. (Canceled).